Stable formulations of polypeptides and uses thereof

ABSTRACT

Formulations are provided that contain single variable domains with a good solubility and good stability under different storage, transportation and stress conditions. The formulations are useful as pharmaceutical formulation. The formulation comprises an aqueous carrier with a pH of 5.5 to 8.0, a buffer selected from the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0; an excipient; and/or a surfactant selected from polysorbate 80, polysorbate 20 and poloxamers. The formulation is further characterized that it has an inorganic salt concentration of 150 mM or lower. The invention further relates to containers and pharmaceutical units comprising such formulations and to methods for preparing and prophylactic and therapeutic uses of the formulations and pharmaceutical units of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/964,469, filed Aug. 12, 2013, which is a continuation-in-partapplication of U.S. patent application Ser. No. 13/393,636, filed Apr.24, 2012, which is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/EP2010/062975, filed Sep. 3, 2010, whichclaims the benefit under 35 U.S.C. §119(e) of U.S. provisionalapplication Ser. No. 61/275,816, filed Sep. 3, 2009 and U.S. provisionalapplication Ser. No. 61/284,502, filed Dec. 18, 2009. U.S. patentapplication Ser. No. 13/964,469 also is a continuation-in-part of U.S.patent application Ser. No. 13/254,266, filed Dec. 9, 2011, which is anational stage filing under 35 U.S.C. §371 of international applicationPCT/EP2010/052600, filed Mar. 2, 2010, which claims the benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 61/284,502,filed Dec. 18, 2009, U.S. provisional application Ser. No. 61/275,816,filed Sep. 3, 2009 and U.S. provisional application Ser. No. 61/157,688,filed Mar. 5, 2009. The disclosures of all of the foregoing relatedapplications are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to formulations of single variabledomains. More specifically the present invention provides formulationsthat contain single variable domains with a good solubility and goodstability under different storage and stress conditions. Theformulations of the invention are suitable for administration to humansubjects.

The invention further relates to containers and pharmaceutical unitscomprising such formulations and to prophylactic and therapeutic uses ofthe formulations and pharmaceutical units of the invention.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

In a broad aspect the present invention generally relates to noveldimer-complexes (herein called “non-fused-dimers” or NFDs) comprisingsingle variable domains such as e.g. Nanobodies®, methods of makingthese complexes and uses thereof. These non-covalently bounddimer-complexes consist of two identical monomers that each comprisesone or more single variable domains (homodimers) or of two differentmonomers that each comprises on or more single variable domains(heterodimers). The subject NFDs have typically altered e.g. improved ordecreased binding characteristics over their monomeric counterpart. TheNFDs of the invention may further be engineered through linkage by aflexible peptide or cysteines in order to improve the stability.

This invention also describes conditions under which such NFDs areformed and conditions under which the formation of such dimers can beavoided. E.g., the present invention also provides methods forsuppressing NFDs such as the dimerization of (human serum)albumin-binding Nanobodies® by adding to a formulation one or moreexcipients that increase the melting temperature of the singe variabledomain such as e.g. by adding mannitol, other polyols or reducing sugarsto a liquid formulation.

The present invention also provides formulations of single variabledomains wherein the formation of NFDs is suppressed. The formulations ofthe invention are suitable for administration to human subjects. Theinvention further relates to containers and pharmaceutical unitscomprising such formulations and to prophylactic and therapeutic uses ofthe formulations and pharmaceutical units of the invention.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND ART

Nanobodies (as further described herein) are characterized by formationof the antigen binding site by a single variable domain, which does notrequire interaction with a further domain (e.g. in the form of VH/VLinteraction) for antigen recognition. Nanobodies have been describedagainst a wide range of different targets (WO 04/062551, WO 05/044858,WO 06/040153, WO 06/122825, WO 07/104529, WO 08/020079, WO 08/074839, WO08/071447, WO 08/074840, WO 08/074867, WO 08/077945, WO 08/101985, WO08/142164, WO 09/068625, WO 08/142165, WO 09/068627) which could beideal candidates for drug development. Nanobodies against IL-6R that caninhibit the IL-6/IL-6R interaction are described in WO 08/020079.Nanobodies against the p19 subunit of IL-23 that block the interactionof IL-23 with its receptor have been described in WO 09/068627.Nanobodies against RANKL that can inhibit osteoclast formation aredescribed in WO 08/142164. The OPG/RANKL/RANK system has recently beendiscovered as pivotal regulatory factors in the pathogenesis of bonediseases and disorders like e.g. osteoporosis.

Proteins, such as therapeutic antibodies and Nanobodies, are oftentransported and/or stored for later use. It is important therefore thatsuch proteins preserve the stability and biological activity of theprotein under various conditions such as different temperature regimensand mechanical stress.

Certain prior liquid antibody preparations have shown short shelf livesand loss of biological activity of the antibodies resulting fromchemical and/or physical instabilities during the transportation andstorage. Chemical instability may be caused by deamidation,racemization, hydrolysis, oxidation, beta elimination or disulfideexchange, and physical instability may be caused by antibodydenaturation, aggregation, precipitation or adsorption. Among those,aggregation, deamidation and oxidation are known to be the most commoncauses of the antibody degradation (Cleland et al., 1993, CriticalReviews in Therapeutic Drug Carrier Systems 10: 307-377). Little isknown about drug formulation components that provide stable liquidformulations of Nanobodies.

There exists a need for stable liquid formulations of Nanobodies whichshow a good solubility of the Nanobody and which exhibit increasesstability, low to undetectable levels of aggregation, low toundetectable levels of Nanobody degradation, and very little to no lossof biological activity of the Nanobody, even under differenttransportation and storage conditions.

The antigen binding sites of conventional antibodies are formedprimarily by the hypervariable loops from both the heavy and the lightchain variable domains. Functional antigen binding sites can howeveralso be formed by heavy chain variable domains (VH) alone. In vivo, suchbinding sites have evolved in camels and camelids as part of heavy chainantibodies, which consist only of two heavy chains and lack lightchains. Furthermore, analysis of the differences in amino acid sequencebetween the VHs of these camel heavy chain-only antibodies (alsoreferred to as VHH) and VH domains from conventional human antibodieshelped to design altered human VH domains (Lutz Riechmann and SergeMuyldermans, J. of Immunological Methods, Vol. 231, Issues 1 to 2, 1999,25-38).

Similarly, it has been shown that by mutation studies of the interfaceresidues as well as of the CDR3 on the VH of the anti-Her2 antibody 4D5in parallel with the anti-hCG VHH H14, some mutations were found topromote autonomous VH domain behaviour (i.e. beneficial solubility andreversible refolding) (Barthelemy P A et al., 2008, J. of Biol.Chemistry, Vol 283, No 6, pp 3639-3654). It was also found thatincreasing the hydrophilicity of the former light chain interface byreplacing exposed hydrophobic residues by more hydrophilic residuesimproves the autonomous VH domain behaviour. These engineered VHs wereshown to be predominantly monomeric at high concentration, however lowquantities of dimers and other aggregates of said engineered VHs werealso found that presumably form relative weak interaction similar tothose described in the art for VL-VH pair interactions. Similarly, acamelized VH, called cVH-E2, is claimed to form dimers in solution in aconcentration dependent manner i.e. at concentrations above 7 mg/ml (butnote that data has not been shown in study; Dottorini et al.,Biochemistry, 2004, 43, 622-628). Below this concentration, the dimerlikely dissociates into monomers and it remains unclear whether thesedimers were active (i.e. binding antigen).

Furthermore, it has recently been reported that a truncated llamaderived VHH (the first seven amino acids are cleaved off) with a veryshort CDR3 (only 6 residues) called VHH-R9 forms a domain swapped dimerin the crystal structure. Since VHH-R9 has been shown to be functionalin solution (low Kd against hapten) and to consist of a monomer only, itis likely that dimerization occurred during the very slowcrystallization process (4 to 5 weeks) and that elements such asN-terminal cleavage, high concentration conditions and short CDR3 couldlead or contribute to the “condensation” phenomena (see in particularalso conclusion part of Spinelli et al., FEBS Letter 564, 2004, 35-40).Sepulveda et al. (J. Mol. Biol. (2003) 333, 355-365) has found thatspontaneous formation of VH dimers (VHD) is in many cases permissive,producing molecules with antigen binding specificity. However, based onthe reported spontaneous formation (versus the dimers formed by PIAreported herein) and the lack of stability data on the non-fused dimers,it is likely that these are weakly interacting dimers similar to theones described by Barthelemy (supra).

Taken together, the literature describes the formation of dimers ofsingle variable domains and fragments thereof that a) are interactingprimarily on relatively weak hydrophobic interaction (which are e.g.depending on the concentration, reversible), and/or b) occur in anotheroccasion only in the crystallisation process (e.g. as a result ofcrystal packing forces). Moreover, it has been described that thesedimers were not binding antigens anymore (as in Spinelli (supra)) or itis unclear whether these dimers were binding dimers (as in Dottorini(supra) and Barthelemy (supra)).

It has been found (see e.g. WO 09/109635) that stable dimer-complexescan be formed in solution with polypeptides comprising at least onesingle variable VHH domain. These dimer-complexes are also hereinreferred to as non-fused-dimers.

SUMMARY OF THE INVENTION

The present invention provides improved formulations (also referred toas “formulation(s) of the invention”) of polypeptides comprising one ormore single variable domains that show good solubility of the singlevariable domains and retain increased stability of the single variabledomains under a variety of different transportation, storage and in-useconditions. The present invention is based on the finding that thepresence in the formulation of certain buffers, certain excipientsand/or certain surfactants may increase the solubility, the meltingtemperature and/or the stability of the single variable domains presentin the formulation.

The present invention provides solubility data for formulations withpolypeptides comprising one or more single variable domains (alsoreferred to as “polypeptide(s) of the invention”) up to 150 mg/mL andhigher. The invention further shows that such formulations can betransported, manipulated through various administration devices andretain activity, purity and potency under different stress conditions:mechanical stress conditions; storage of the formulation at variousstress conditions such as different freeze/thaw cycles, at 2-8° C., at25±5° C. and at elevated temperature.

The formulation of the present invention comprises an aqueous carrierwith a pH of 5.5 to 8.0 and a polypeptide comprising one or more singlevariable domains at a concentration of 1 mg/ml to 200 mg/ml, saidformulation being formulated for administration to a human subject andsaid formulation further comprising one or more components selectedfrom:

-   -   a) A buffer at a concentration of 10 mM to 100 mM selected from        the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0,        MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;    -   b) An excipient at a concentration of 1% to 20% (w:v);    -   c) A surfactant at a concentration of 0.001% to 1% selected from        TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 and poloxamers;        wherein said formulation has an inorganic salt concentration of        150 mM or lower.

In a preferred aspect, the formulation of the invention comprises atleast two of the above components, such as e.g. at least the componentsin a) and b), at least the components in a) and c) or at least thecomponents in b) and c). Preferably, the formulation of the inventioncomprises the components in a), b) and c).

The present inventors observed that formulations that have 150 mM orless of inorganic salt show a much better stability of the singlevariable domains contained in the formulation. Inorganic saltsfrequently used in pharmaceutical formulation are NaCl and KCl. Thesingle variable domains present in formulations containing 150 mM orless of inorganic salt have shown increased stability (e.g. lesstendency to form aggregates, dimers and/or pyroglutamate, or to loosepotency) at different stress storage conditions (such as e.g. duringstorage at a temperature of 37±5° C. up to at least 2 weeks (preferablyat least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks,at least 3 months, at least 6 months, at least 1 year, 1.5 year or even2 years or more)), an improved melting temperature and/or an increasedsolubility. Preferably, the formulation contains 100 mM or less ofinorganic salt, more preferably even 50 mM or less of inorganic salt.Most preferably the formulation does not contain any inorganic salt.

The polypeptide (also referred to as “polypeptide of the invention”)comprising one or more single variable domains for use in theformulation of the invention may be therapeutic or prophylactic, and maybe useful in the prevention, treatment and/or management of one or morediseases and/or disorders. In one specific aspect, the polypeptide hasat least two single variable domains. In another specific aspect, thepolypeptide has at least three single variable domains. Preferredpolypeptides of the invention and single variable domains used in thepolypeptide of the invention are described in WO 08/142164, WO 08/020079and WO 09/068627. Particularly preferred polypeptides of the inventionmay be selected from SEQ ID NO's: 1 to 6.

The polypeptide of the invention may be present in the formulation ofthe present invention at a concentration of about 1 to 200 mg/mL ormore, preferably about 5 to 100 mg/mL or more, more preferably about 5to 50 mg/mL or more, most preferably about 5 to 30 mg/mL or more, suchas around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30 mg/mL,around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70 mg/mL,around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, around 150 mg/mL oreven more.

In an aspect of the invention, the formulation is homogeneous. Inanother aspect, the formulation of the invention is sterile. In additionto the polypeptide of the invention, the formulation of the presentinvention comprises at least an aqueous carrier (e.g. distilled water,MILLI-Q water or WFI) and a buffer.

The pH of the formulation of the invention should be in the range of 5.5to 8.0, preferably the pH is around 6.0 to 7.5, more preferably around6.2 to 7.5 or around 6.2 to 7.0. Most preferably the pH is in the rangeof 6.5 to 7.0, such as e.g. pH 6.5. These pH ranges have shown toprovide an increased melting temperature to the polypeptides present inthe formulation of the invention.

Preferred buffers for use in the formulation of the invention are hepespH 7.0-8.0, histidine pH 6.0-6.5, MES pH 6.0, succinate pH 6.0-6.5 oracetate pH 5.5-6.0, preferably hepes pH 7.0 or histidine pH 6.0-6.5,most preferably histidine pH 6.0, 6.2 or 6.5. Formulations comprisingone of these buffers have shown a very good solubility (as definedherein) of the polypeptides of the invention, an improved meltingtemperature of the polypeptides present in the formulation and increasedstability (e.g. less tendency to form aggregates, dimers and/orpyroglutamate, or to lose potency) at different stress storageconditions (such as e.g. during storage at a temperature of 37±5° C. upto at least 2 weeks (preferably at least 3 weeks, at least 5 weeks, atleast 8 weeks, at least 10 weeks, at least 3 months, at least 6 months,at least 1 year, 1.5 year or even 2 years or more)). The buffer ispreferably at a concentration of about 10 to 20 mM, such as 10 mM or 15mM. In a specific aspect, the formulation of the invention comprises ahistidine buffer pH 6.5 at a concentration of 15 mM or a histidinebuffer pH 6.0 at a concentration of 10 mM.

Accordingly, the present invention provides stable formulations ofpolypeptides comprising one or more single variable domains, saidformulations comprising an aqueous carrier, the polypeptide at aconcentration from about 1 to 200 mg/mL or more, preferably about 5 to100 mg/mL or more, more preferably about 5 to 50 mg/mL or more, mostpreferably about 5 to 30 mg/mL or more, such as around 5 mg/mL, around10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40 mg/mL, around 50mg/mL, around 60 mg/mL, around 70 mg/mL, around 80 mg/mL, around 90mg/mL, around 100 mg/mL, around 150 mg/mL or even more, and a buffersuch as a histidine buffer with a pH ranging from 6.0 to 7.0 at aconcentration of about 10 to 20 mM.

Preferably the formulation of the invention is isotonic or slightlyhypotonic and/or has an osmolality of about 290±60 mOsm/kg, such asabout 240 or higher, 250 or higher or 260 or higher. Isotonicity of theformulation can be further adjusted by the addition of one or moreexcipients and/or tonifiers.

Preferred excipients/tonifiers for use in the formulation of the presentinvention are saccharides and/or polyols. Accordingly, in anotheraspect, the formulation of the invention comprises a saccharide and/orpolyol. Formulations comprising one or more saccharides and/or polyolshave shown increased stability (e.g. less tendency to form aggregates,dimers and/or pyroglutamate, or to loose potency) at different stressstorage conditions (such as e.g. during storage at a temperature of37±5° C. up to at least 2 weeks (preferably at least 3 weeks, at least 5weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at least6 months, at least 1 year, 1.5 year or even 2 years or more) and duringmechanical stress conditions) and/or an improved melting temperature ofthe polypeptides present in the formulation. In a specific aspect of theinvention, the excipient present in the formulation of the invention isa non-reducing sugar. In another specific aspect, the excipient presentin the formulation of the invention is a disaccharide. In anotherspecific aspect, the excipient present in the formulation of theinvention is selected from sucrose, trehalose, sorbitol and mannitol.The saccharide and/or polyol is preferably present in the formulation ofthe invention at a concentration of about 1% to 20%, preferably about2.5% to 15%, more preferably about 5% to 10%, such as around 5%, around7.5%, around 8% or around 10%.

Accordingly, the present invention provides stable formulations ofpolypeptides comprising one or more single variable domains, saidformulations comprising an aqueous carrier, the polypeptide at aconcentration from about 1 to 200 mg/mL or more, preferably about 5 to100 mg/mL or more, more preferably about 5 to 50 mg/mL or more, mostpreferably about 5 to 30 mg/mL or more, such as around 5 mg/mL, around10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40 mg/mL, around 50mg/mL, around 60 mg/mL, around 70 mg/mL, around 80 mg/mL, around 90mg/mL, around 100 mg/mL, around 150 mg/mL or even more, and a saccharideand/or polyol at a concentration of about 1% to 20%, preferably about2.5% to 15%, more preferably about 5% to 10%, such as around 5%, around7.5%, around 8% or around 10%.

In another specific aspect, the formulation of the invention maycomprise one or more surfactants (e.g., TWEEN (polysorbate) 20, TWEEN(polysorbate) 80 or a poloxamer). Formulations comprising a surfactanthave shown a very good solubility (as defined herein) of thepolypeptides of the invention and/or increased stability undermechanical stress. The surfactant may be present at a concentration inthe range of about 0.001% to 1% (preferably between about 0.001% to0.1%, or about 0.01% to 0.1% such as around 0.001%, around 0.005%,around 0.01%, around 0.02%, around 0.05%, around 0.08%, around 0.1%,around 0.5%, or around 1% of the formulation, preferably around 0.01%).

Accordingly, the present invention provides stable formulations ofpolypeptides comprising one or more single variable domains, saidformulations comprising an aqueous carrier, the polypeptide at aconcentration from about 1 to 200 mg/mL or more, preferably about 5 to100 mg/mL or more, more preferably about 5 to 50 mg/mL or more, mostpreferably about 5 to 30 mg/mL or more, such as around 5 mg/mL, around10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40 mg/mL, around 50mg/mL, around 60 mg/mL, around 70 mg/mL, around 80 mg/mL, around 90mg/mL, around 100 mg/mL, around 150 mg/mL or even more, and a surfactant(e.g., TWEEN (polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer) ata concentration in the range of about 0.001% to 1% (preferably betweenabout 0.001% to 0.1%, or about 0.01% to 0.1% such as around 0.001%,around 0.005%, around 0.01%, around 0.02%, around 0.05%, around 0.08%,around 0.1%, around 0.5%, or around 1% of the formulation, preferablyaround 0.01%).

A preferred formulation of the invention may comprise:

a) A histidine pH 6.5 buffer at a concentration of 10 mM to 100 mM, suchas 10 mM to 20 mM;

b) Sucrose at a concentration of 1% to 10%; and

c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.

Another preferred formulation of the invention may comprise:

a) A histidine pH 6.5 buffer at a concentration of 15 mM;

b) Sucrose at a concentration of 8%; and

c) TWEEN (polysorbate) 80 at a concentration of 0.01%.

Another preferred formulation of the invention may comprise:

a) A histidine pH 6.0 buffer at a concentration of 10 mM to 100 mM, suchas 10 mM to 20 mM;

b) Sucrose at a concentration of 1% to 10%; and

c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.

Another preferred formulation of the invention may comprise:

a) A histidine pH 6.0 buffer at a concentration of 10 mM;

b) Sucrose at a concentration of 10%; and

c) TWEEN (polysorbate) 80 at a concentration of 0.005%.

The present invention provides formulations of a polypeptide comprisingone or more single variable domains which exhibit high solubility of thepolypeptide, little to no aggregation of the polypeptide and highstability during long periods of storage.

In one aspect, the components present in the formulations of theinvention have been selected such that the polypeptides of the inventionhave a solubility of at least 20 mg/mL, at least 50 mg/mL, preferably atleast 90 mg/mL, at least 120 mg/mL, at least 150 mg/mL or even 200 mg/mLor more.

In another aspect, the components present in the formulations of theinvention have been selected such that the polypeptide present in theformulation of the invention has a melting temperature of at least 59°C. or more (such as 59.5° C. or more), preferably at least 60° C. ormore (such as 60.5° C. or more), more preferably at least 61° C. or more(such as 61.5° C. or more) or at least 62° C. or more (such as 62.5° C.or more), most preferably at least 63° C. or more (such as 63.5° C. ormore) as measured by the thermal shift assay (TSA) and/or differentialscanning calorimetry (DSC).

In yet another aspect, the formulation of the present invention exhibitsstability under various stress conditions such as:

-   -   multiple (up to 10) freeze/thaw cycles;    -   storage at a temperature of 2-8° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   storage at a temperature of 25±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   storage at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more); and/or    -   mechanical stress.

Mechanical stress as used in the present invention can be any form ofexternal force applied on the formulation that may affect the stabilityof the polypeptide present in the formulation. Without being limiting,the mechanical stress applied to the solution can be shear stress, stirstress, shake stress, rotation stress, etc. Preferably the formulationof the invention is stable under one or more of the following forms ofmechanical stress:

-   -   shaking the formulation during 10 s to 1 min;    -   pushing the formulation through a needle (25G, preferably 26G,        more preferably 27G, even more preferably 28G, most preferably        29G or more) with a syringe (the syringe used can be any        commercially available syringe, such as e.g. a 1 mL, 2 mL, 3 mL,        4 mL, 5 ml, 10 mL up to 50 mL syringe);    -   rotating for two days at 10 rpm; and/or    -   stirring for 1 hour at room temperature and/or 4-48 hours (such        as 4-8 hours, 12 hours, 24 hours or even 48 hours) at 4° C. at        at least 10 rpm (such as 50 rpm, 100 rpm or more).

Preferably, the formulations of the present invention are stable undermore than one (such as two, three, four, five, six or seven) of theabove stress conditions, most preferably under all of the above stressconditions.

Accordingly, the polypeptide of the invention present in the formulationof the invention:

-   -   is stable after multiple (up to 10) freeze/thaw cycles, said        stability as determined by OD320/OD280 measurement, SE-HPLC,        RP-HPLC, IEX-HPLC, potency assay (such as BIACORE or ELISA)        and/or SDS-PAGE;    -   is stable during storage at a temperature of 2-8° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more), said        stability as determined by OD320/OD280 measurement, SE-HPLC,        RP-HPLC, IEX-HPLC, potency assay (such as BIACORE or ELISA)        and/or SDS-PAGE;    -   is stable during storage at a temperature of 25±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more), said        stability as determined by OD320/OD280 measurement, SE-HPLC,        RP-HPLC, IEX-HPLC, potency assay (such as BIACORE or ELISA)        and/or SDS-PAGE;    -   is stable during storage at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more), said        stability as determined by OD320/OD280 measurement, SE-HPLC,        RP-HPLC, IEX-HPLC, potency assay (such as BIACORE or ELISA)        and/or SDS-PAGE;    -   is stable when shaking the formulation during 10 s to 1 min;    -   is stable when pushing the formulation through a needle (25G,        preferably 26G, more preferably 27G, even more preferably 28G,        most preferably 29G or more) with a syringe (the syringe used        can be any commercially available syringe, such as e.g. a 1 mL,        2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL up to 50 mL        syringe);    -   is stable when rotating for two days at 10 rpm; and/or    -   is stable when stirring for 1 hour at room temperature and/or        4-48 hours (such as 4-8 hours, 12 hours, 24 hours or even 48        hours) at 4° C. at at least 10 rpm (such as 50 rpm, 100 rpm or        more).

The stability of the formulations of the present invention can bedemonstrated by the fact that less than 10% of the polypeptides formspyroglutamate at the N-terminal glutamic acid (e.g. as assessed byRP-HPLC) and/or less than 10% of the polypeptides forms dimers (e.g. asassessed by SE-HPLC) during storage under one or more of the abovestress conditions. Preferably less than 10% of the polypeptides formspyroglutamate at the N-terminal glutamic acid (e.g. as assessed byRP-HPLC) and less than 10% of the polypeptides forms dimers (e.g. asassessed by SE-HPLC) during storage under one or more of the abovestress conditions.

In a specific aspect, less than 10% of the polypeptides present in theformulation of the invention forms pyroglutamate at the N-terminalglutamic acid (e.g. as assessed by RP-HPLC) during storage at atemperature of 37±5° C. for up to at least 2 weeks (preferably at least3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least3 months, at least 6 months, at least 1 year, 1.5 year or even 2 yearsor more). In another specific aspect, less than 10% of the polypeptidesforms dimers (e.g. as assessed by SE-HPLC) during storage at atemperature of 37±5° C. for up to at least 2 weeks (preferably at least3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least3 months, at least 6 months, at least 1 year, 1.5 year or even 2 yearsor more). In yet another specific aspect, less than 10% of thepolypeptides present in the formulation of the invention formspyroglutamate at the N-terminal glutamic acid (e.g. as assessed byRP-HPLC) during storage at a temperature of 37±5° C. for up to at least2 weeks (preferably at least 3 weeks, at least 5 weeks, at least 8weeks, at least 10 weeks, at least 3 months, at least 6 months, at least1 year, 1.5 year or even 2 years or more) and less than 10% of thepolypeptides forms dimers (e.g. as assessed by SE-HPLC) during storageat a temperature of 37±5° C. for up to at least 2 weeks (preferably atleast 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, atleast 3 months, at least 6 months, at least 1 year, 1.5 year or even 2years or more).

Apart from this and/or in addition, the stability of the formulations ofthe present invention can be demonstrated by the fact that it shows onlylow to undetectable levels of aggregation and/or particulate formation(e.g. as assessed by SE-HPLC, subvisible particle counting, analyticalultracentrifugation, dynamic light scattering, OD320/OD280 ratiomeasurement and/or elastic light scattering) even during storage underone ore more of the above stress conditions. In a specific aspect, theformulations of the present invention show only low to undetectablelevels of aggregation and/or particulate formation (e.g. as assessed bySE-HPLC, subvisible particle counting, analytical ultracentrifugation,dynamic light scattering and/or OD320/OD280 measurement) at atemperature of 37±5° C. and/or 5±5° C. for up to at least 2 weeks(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks, atleast 10 weeks, at least 3 months, at least 6 months, at least 1 year,1.5 year or even 2 years or more).

Apart from this and/or in addition, the stability of the formulations ofthe present invention can be demonstrated by the fact that it shows onlylow to undetectable levels of fragmentation and/or degradation of thepolypeptides (e.g. as assessed by SDS-PAGE, SE-HPLC, RP-HPLC and/orIEX-HPLC) even during storage under one or more of the above stressconditions. In a specific aspect, the formulations of the presentinvention show only low to undetectable levels of fragmentation and/ordegradation of the polypeptides (e.g. as assessed by SDS-PAGE, SE-HPLC,RP-HPLC and/or IEX-HPLC) at a temperature of 37±5° C. for up to at least2 weeks (preferably at least 3 weeks, at least 5 weeks, at least 8weeks, at least 10 weeks, at least 3 months, at least 6 months, at least1 year, 1.5 year or even 2 years or more).

Apart from this and/or in addition, the stability of the formulations ofthe present invention can be demonstrated by the fact that it shows verylittle to no loss of the biological activities of the polypeptide of theinvention (e.g. as assessed by ELISA and/or BIACORE) even during storageunder one or more of the above stress conditions. In a specific aspect,the formulations of the present invention show very little to no loss ofthe biological activities of the polypeptide of the invention (e.g. asassessed by ELISA and/or BIACORE) at a temperature of 37±5° C. for up toat least 2 weeks (preferably at least 3 weeks, at least 5 weeks, atleast 8 weeks, at least 10 weeks, at least 3 months, at least 6 months,at least 1 year, 1.5 year or even 2 years or more).

More specifically, in the formulations of the present invention at least80% (preferably at least 90%, more preferably at least 95% or even atleast 99%) of the polypeptides retain their binding activity to at leastone (preferably to all) of their targets (e.g. as assessed by ELISAand/or BIACORE) after storage under one or more of the above stressconditions compared to the binding activity prior to storage.

In a specific aspect, at least 80% (preferably at least 90%, morepreferably at least 95% or even at least 99%) of the polypeptidesretains their binding activity (e.g. as assessed by ELISA and/orBIACORE) to at least one (preferably to all) of their targets afterstorage at 37±5° C. for up to at least 2 weeks (preferably at least 3weeks, at least 5 weeks, at least 2 months, at least 6 months, at least1 year, 1.5 year or even 2 years or more) compared to the bindingactivity prior to storage.

Accordingly the present invention provides stable formulations ofpolypeptides comprising one or more single variable domains, wherein:

-   -   less than 10% of the polypeptides forms pyroglutamate at the        N-terminal glutamic acid (e.g. as assessed by RP-HPLC) during        storage at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   less than 10% of the polypeptides forms dimers (e.g. as assessed        by SE-HPLC) during storage at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more);    -   at least 80% of the polypeptides retain its binding activity        (e.g. as assessed by ELISA and/or BIACORE) to at least one        (preferably to all) of its targets after storage at 37±5° C. up        to 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 2 months, at least 6 months, at least 1 year, 1.5 year or        even 2 years or more) compared to the binding activity prior to        storage; and/or    -   the polypeptide is stable under mechanical stress.

In a preferred aspect, the formulation of the invention is apharmaceutical formulation.

The present invention further provides methods for preparing the stableformulations of the invention. The methods of the invention may comprisethe steps of concentrating a polypeptide comprising one or more singlevariable domains and exchanging it with the preferred buffer and/orexcipient.

Also provided are containers, kits and pharmaceutical unit dosagescomprising the formulations of the invention for use by, e.g., ahealthcare professional. In specific embodiments, the kits orpharmaceutical unit dosages comprising the stable formulations of theinvention are formulated for parenteral administration (e.g.,intradermally, intramuscularly, intraperitoneally, intravenously and/orsubcutaneously) of the polypeptide of the invention to a human subject.The formulations, containers, pharmaceutical unit dosages and/or kitscan be used in prophylaxis and/or therapy. In a specific aspect, theformulations, containers, pharmaceutical unit dosages and/or kits areused for the prevention and/or treatment of one ore more diseases and/ordisorders such as bone diseases and/or disorders (such as e.g.osteoporosis, cancer-related bone diseases, and/or bone loss associatedwith autoimmunity and/or viral infection) or autoimmune diseases (suchas e.g. rheumatoid arthritis).

The present invention provides methods and formulations that avoid theformation of dimer-complexes of single variable domains. In one aspectthe present invention provides a formulation (also referred to herein as“formulation of the invention”), such as a pharmaceutical formulation,comprising i) a polypeptide that comprises at least one single variabledomain, and ii) an excipient, preferably selected from a polyol, anon-reducing sugar and/or a dissaccharide. Preferred excipients for usein the formulation of the invention include sorbitol, mannitol, xylitol,ribitol, trehalose, sucrose and/or erythritol. The excipient ispreferably present at a concentration of 1% to 20%, 2.5% to 15%,preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%.

The present inventors have shown that the addition of such an excipientin a formulation can drastically reduce the formation of non-fuseddimers of single variable domains. The formulation of the invention istherefore particularly suitable for use with polypeptides comprising atleast one single variable domain, wherein said single variable domain issusceptible to dimerization.

As indicated in the background art, it has been found (see e.g. WO09/109635) that stable dimer-complexes can be formed in solution forpolypeptides comprising at least one single variable VHH domain,preferably for polypeptides comprising at least one single variable VHHdomain that forms dimers using the methods described herein (i.e.process-induced association, introduction of CDR3/framework region 4destabilizing residues and/or storage at high temperature and highconcentration), more preferably for polypeptides comprising at least onesingle variable VHH domain with sequences SEQ ID NO: 7 to 12 and 17-20and/or variants thereof, e.g. single variable VHH domain with sequencesthat are 70% and more identical to SEQ ID NO: 7 to 12 and 17-20. Some ofthese stable dimer-complexes (also herein referred to asnon-fused-dimers or NFDs; non-fused-dimer or NFD) can retain bindingfunctionality to at least 50% or can even have increased bindingaffinity compared to their monomeric building blocks, others havedecreased or no binding functionality anymore. These NFDs are much morestable compared to the ‘transient’ concentration-dependent dimersdescribed e.g. in Barthelemy (supra) and are once formed stable in awide range of concentrations. These NFDs may be formed by swappingframework 4 region between the monomeric building blocks whereby bothsaid monomeric building blocks interlock (see experimental part of thecrystal structure of polypeptide B NFD). These dimers are typicallyformed upon process-induced association (PIA) using methods describedherein and/or storage at relative high temperature over weeks (such ase.g. 37° C. over 4 weeks) and high concentration (such as e.g. higherthan 50 mg/ml, e.g. 65 mg/ml).

As indicated above, the invention teaches methods and formulations thatavoid the formation of such dimer-complexes in i) e.g. an up-scaledproduction or purification process of said polypeptides comprisingsingle variable domain(s) under non-stress condition (i.e. conditionthat do not favour unfolding of immunoglobulins), ii) by an adequateformulation with excipients increasing the melting temperature of thesingle variable domain(s), e.g. by having mannitol in the formulationand/or iii) by increasing the stability of the CDR3 and/or framework 4region conformation.

Accordingly, in one aspect, the present invention relates to aformulation that comprises a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, and further comprising an excipient at aconcentration of 1% to 20% (w:v). Preferred excipients for use in theformulation of the present invention are saccharides and/or polyols.Accordingly, in another aspect, the formulation of the inventioncomprises a saccharide and/or polyol. Formulations comprising one ormore saccharides and/or polyols have shown increased stability (i.e.less tendency to form dimmers and/or oligomers and/or or to losepotency) at different stress storage conditions (such as e.g. duringstorage at a temperature of 37±5° C. up to at least 2 weeks (preferablyat least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks,at least 3 months, at least 6 months, at least 1 year, 1.5 year or even2 years or more)) and/or an improved melting temperature of thepolypeptides present in the formulation. In a specific aspect of theinvention, the excipient present in the formulation of the invention isa non-reducing sugar. In another specific aspect, the excipient presentin the formulation of the invention is a disaccharide. In anotherspecific aspect, the excipient present in the formulation of theinvention is selected from sucrose, trehalose, sorbitol and mannitol.The saccharide and/or polyol is preferably present in the formulation ofthe invention at a concentration of about 1% to 20%, preferably about2.5% to 15%, more preferably about 5% to 10%, such as around 5%, around7.5%, around 8% or around 10%.

The stability of the formulations of the present invention can bedemonstrated by the fact that they show only low to undetectable levelsof dimer and/or oligomer formation (e.g. as assessed by SE-HPLC) evenduring storage under one or more stress conditions, such as at atemperature of 37±5° C. and/or 5±5° C. for up to at least 2 weeks(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks, atleast 10 weeks, at least 3 months, at least 6 months, at least 1 year,1.5 year or even 2 years or more). The stability of the formulations ofthe present invention can also be demonstrated by the fact that theyshow very little to no loss of the biological activities (e.g. asassessed by ELISA and/or BIACORE) even during storage under one or morestress conditions, such as at a temperature of 37±5° C. for up to atleast 2 weeks (preferably at least 3 weeks, at least 5 weeks, at least 8weeks, at least 10 weeks, at least 3 months, at least 6 months, at least1 year, 1.5 year or even 2 years or more).

More specifically, in the formulations of the present invention at least80% (preferably at least 90%, more preferably at least 95% or even atleast 99%) of the polypeptides retains its binding activity to at leastone (preferably to all) of its targets (e.g. as assessed by ELISA and/orBIACORE) after storage under one or more of the above stress conditionscompared to the binding activity prior to storage. In a specific aspect,at least 80% (preferably at least 90%, more preferably at least 95% oreven at least 99%) of the polypeptides retains its binding activity(e.g. as assessed by ELISA and/or BIACORE) to at least one (preferablyto all) of its targets after storage at 37±5° C. for up to at least 2weeks (preferably at least 3 weeks, at least 5 weeks, at least 2 months,at least 6 months, at least 1 year, 1.5 year or even 2 years or more)compared to the binding activity prior to storage.

Accordingly the present invention provides stable formulations ofpolypeptides comprising one or more single variable domains, wherein:

-   -   less than 10% of the polypeptides forms dimers (e.g. as assessed        by SE-HPLC) during storage at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more);    -   at least 80% of the polypeptides retain its binding activity        (e.g. as assessed by ELISA and/or BIACORE) to at least one        (preferably to all) of its targets after storage at 37±5° C. up        to 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 2 months, at least 6 months, at least 1 year, 1.5 year or        even 2 years or more) compared to the binding activity prior to        storage; and/or

The present invention further provides methods for preparing the stableformulations of the invention. The methods of the invention may comprisethe steps of concentrating a polypeptide comprising one or more singlevariable domains and exchanging it with the preferred buffer and/orexcipient.

Also provided are containers, kits and pharmaceutical unit dosagescomprising the formulations of the invention for use by, e.g., ahealthcare professional. In specific embodiments, the kits orpharmaceutical unit dosages comprising the stable formulations of theinvention are formulated for parenteral administration (e.g.,intradermally, intramuscularly, intraperitoneally, intravenously and/orsubcutaneously) of the polypeptide of the invention to a human subject.The formulations, containers, pharmaceutical unit dosages and/or kitscan be used in prophylaxis and/or therapy. In a specific aspect, theformulations, containers, pharmaceutical unit dosages and/or kits areused for the prevention and/or treatment of one or more diseases and/ordisorders such as vascular diseases and/or disorders (such as e.g. acutecoronary syndrome (ACS), myocardial infarction, thromboticthrombocytopenic purpura (TTP) or Moschcowitz syndrome, vascularsurgery, stroke), bone diseases and/or disorders (such as e.g.osteoporosis, cancer-related bone diseases, and/or bone loss associatedwith autoimmunity and/or viral infection) or autoimmune diseases (suchas e.g. rheumatoid arthritis).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) and FIG. 1(B). The 280 nm SE-HPLC chromatograms of RANKL008aformulated in phosphate (FIG. 1(A)), or histidine (FIG. 1(B)) bufferswith either 50 mM NaCl, 100 mM NaCl or 10% mannitol, before and after 10freeze/thaw cycles. A zoom on the main peak is shown as inset.

FIG. 2. The 280 nm RP-HPLC chromatograms of RANKL008a formulated inphosphate buffers with either 50 mM NaCl, 100 mM NaCl or 10% mannitol,before and after 10 freeze/thaw cycles. A zoom on the main peak is shownas inset.

FIG. 3. The 280 nm SE-HPLC chromatograms of RANKL008a formulated inphosphate buffer with either 50 mM NaCl, 100 mM NaCl or 10% mannitol,after incubation for 2 weeks at 37° C. A zoom on the main peak is shownas inset.

FIG. 4(A) and FIG. 4(B). Figure demonstrating the time-dependentdecrease (FIG. 4(A)) and increase (FIG. 4(B)) of the surface area of,respectively, the main peak (FIG. 4(A)) and % dimers (FIG. 4(B))observed in SE-HPLC analysis of RANKL008a formulated in differentbuffers and stored up to 10 weeks at 37° C.

FIG. 5. The 280 nm RP-HPLC chromatograms of RANKL008a formulated inphosphate buffer with either 50 mM NaCl, 100 mM NaCl or 10% mannitol,after incubation for 2 weeks at 37° C. A zoom on the main peak is shownas inset.

FIG. 6. Overlay of the 280 nm IEX-HPLC chromatograms of RANKL008aformulated in phosphate buffer with either 50 mM NaCl, 100 mM NaCl or10% mannitol, after incubation for 2 weeks at 37° C. A zoom on the mainpeak and postpeaks is shown as inset.

FIG. 7. Relative amounts (%) of the main peak and the two postpeaksobserved in the IEX-HPLC chromatograms of the stability samples afterstorage for 10 weeks at 37° C.

FIG. 8. Picture and visual observation of the vials after shaking. TheRANKL008a sample, diluted (to 5 mg/mL) and undiluted with or without0.01% TWEEN (polysorbate) 80 was shaken strongly (10 s-1 min).

FIG. 9. Overview of dilutions and steps made in syringeability study asdescribed in Example 1.6.

FIG. 10. OD 320/278 and OD 350/278 ratios (n=3) after passage/storage ofRANKL008a in syringes with different diluents as described in Example1.6.

FIG. 11. Relative HSA and RANKL potency of RANKL008a after dilution indifferent diluents and passage/storage in syringes as described inExample 1.6.

FIG. 12. Overview of needle/gauge size study with diluted RANKL008a asdescribed in Example 1.7.

FIG. 13. Overview of further needle/gauge size study with diluted andundiluted RANKL008a as described in Example 1.7.

FIG. 14(A), FIG. 14(B), FIG. 14(C), FIG. 14(D), FIG. 14(E) and FIG.14(F). Overview of the results obtained for thermal stability testing ofIL6R304 (FIG. 14(A), FIG. 14(C), FIG. 14(E)) and IL6R305 (FIG. 14(B),FIG. 14(D), FIG. 14(F)) and in function of NaCl concentration (FIG.14(A), FIG. 14(B)), mannitol added (FIG. 14(C), FIG. 14(D) andbuffer/excipient (FIG. 14(E), FIG. 14(F)).

FIG. 15(A). Overview of the results obtained in thermal stabilitytesting of IL6R304 in Tris buffer pH 7.2 or Histidine pH 6.5, withsucrose, glycine or mannitol added as excipient.

FIG. 15(B). Overview of the results obtained in thermal stabilitytesting of IL6R304 in Tris buffer pH 7.2 or Histidine pH 6.5, withseveral concentrations of NaCl added.

FIG. 15(C). Overview of the results obtained in thermal stabilitytesting of IL6R304 in Tris buffer pH 7.2 or Histidine pH 6.5, withseveral concentrations of mannitol added.

FIG. 16(A). Thermograms (obtained after subtracting the base-lines) ofIL6R304 in the buffers as indicated in the graph.

FIG. 16(B). Thermograms (obtained after subtracting the base-lines) ofIL6R304 in the citrate buffer as indicated in the graph.

FIG. 17(A) and FIG. 17(B). PAMAS analysis of IL6R304 formulated in PBScompared to IL6R304 formulated in PBS+0.01% or 0.02% Tween 80. FIG.17(A): Counts of particles: >25 μm, >50 μm, >100 μm and >200 μm. FIG.17(B): Counts of particles: >10 μm, >25 μm, >50 μm, >100 μm and >200 μm.

FIG. 18. SE-HPLC for the IL6R304 molecule in the presence of differentconcentrations of TWEEN (polysorbate) 80.

FIG. 19(A), FIG. 19(B), FIG. 19(C) and FIG. 19(D). (FIG. 19(A))-(FIG.19(C)): Log-values of the soluble IL-6R Nanobody concentration (Y-axis)vs. PEG6000 concentration (%) (X-axis), together with the calculatedsolubility values. (FIG. 19(D)) represents an example of how the linearregression analysis is performed on the obtained data points todetermine the intercept with the X-axis (from which the theoreticalsolubility at zero % PEG6000 can be deduced).

FIG. 20. Overlay of the SE-HPLC chromatograms of IL6R304 formulated at10 mg/mL stored for 3 weeks at 37° C. Inset, zoom on the main peak todemonstrate the buffer-dependent differences in % aggregates.

FIG. 21. Figure demonstrating the buffer-dependent differences in %aggregates (peak surface area in SE-HPLC) that were observed in thestability samples of IL6R304 and IL6R305 stored for 1 week at 37° C.

FIG. 22. Figure demonstrating the buffer-dependent differences in %aggregates (peak surface area in SE-HPLC) that were observed in thestability samples of IL6R304 and IL6R305 stored for 3 weeks at 37° C.

FIG. 23(A), FIG. 23(B), FIG. 23(C) and FIG. 23(D). Overlays of theRP-HPLC chromatograms from IL6R304 incubated for 3 weeks at 37° C. indifferent formulation buffers. A zoom on the main peak is shown asinset. Note the increase of two minor prepeaks and a postpeak in theIL6R304 samples stored for 3 weeks. FIG. 23(A): shows control, PBS andPBS+5% mannitol. FIG. 23(B): shows control, 10 mM phosphate (pH7)+100 mMNaCl, and 10 mM phosphate (pH7)+100 mM NaCl+5% mannitol. FIG. 23(C):shows control, 10 mM acetate (pH5.5)+100 mM NaCl, and 10 mM acetate(pH5.5)+100 mM NaCl+5% mannitol. FIG. 23(D): shows control, 20 mMhistidine (pH6.0)+100 mM NaCl, and 20 mM histidine (pH6.0)+100 mMNaCl+5% mannitol.

FIG. 24. Osmolality data of IL6R304 (10 mg/mL) in the 12 differentbuffer indicated in the graph. The horizontal bar defines the isotonicregion.

FIG. 25(A) and FIG. 25(B). Figure demonstrating the time-dependentincrease of the % oligomers/aggregates (Y-axis) observed in SE-HPLCanalysis of IL6R304 stored for up to 5 weeks at 37° C. (FIG. 25(A)) and5° C. (FIG. 25(B)) in the buffers indicated in the graph. The %oligomers/aggregates is expressed as the sum of the % peak surface areasof prepeak 1a, prepeak 1b and prepeak 2 relative to the total peaksurface area.

FIG. 26(A) and FIG. 26(B). FIG. 26(A): the % oligomers (% peak surfacearea) observed in the Histidine buffers after storage for 3 weeks at 37°C. compared to the equivalent sample in PBS buffer. FIG. 26(B):time-dependent and buffer-dependent increase in the % oligomers observedin the IL-6R stability samples stored for up to 5 weeks at 37° C., at aconcentration of 10 mg/mL in the buffers indicated in the graph.

FIG. 27(A) and FIG. 27(B). SE-HPLC profile of IL6R304 in the differentbuffers as depicted in Table 21. IL6R304 was less prone tooligomerization in L-histidine buffer compared to phosphate buffer. FIG.27(A): Less oligomers, which were seen as prepeaks during SE-HPLCanalysis, were present in IL6R304 samples stored for 8 weeks at 37° C.in L-histidine buffer (buffers 1-5) compared to phosphate buffer(buffers 6-10). The amount of oligomers was lowest in buffer 3. FIG.27(B): The effect of the different excipients on oligomerization ineither L-histidine versus phosphate. Note that the surface area of thepostpeak was higher in phosphate compared to L-histidine.

FIG. 28. Kinetics of oligomer formation upon storage of IL6R304 in thedifferent buffers. Oligomerization was significantly slower inL-histidine (buffers 1-5) compared to phosphate buffer (buffers 6-10).

FIG. 29(A), FIG. 29(B), FIG. 29(C), FIG. 29(D), FIG. 29(E) and FIG.29(F). Overlay of the RP-HPLC chromatograms from IL6R304 after storagefor up to 8 weeks at +37° C. in 10 different formulation buffers. A zoomon the main peak and sidepeaks is shown as inset. FIG. 29(A): Buffers1-5, storage for 1 week. FIG. 29(B): Buffers 6-10, storage for 1 week.FIG. 29(C): Buffers 1-5, storage for 4 weeks. FIG. 29(D): Buffers 6-10,storage for 4 weeks. FIG. 29(E): Buffers 1-5, storage for 8 weeks. FIG.29(F): Buffers 6-10, storage for 8 weeks.

FIG. 30. Kinetics of the formation for the pyroglutamate variant ofIL6R304 upon storage under stressed conditions in the different buffers.Less pyroglutamate is formed in L-histidine compared to phosphatebuffer.

FIG. 31. Aggregation index before and after stirring of IL6R304.

FIG. 32(A) and FIG. 32(B). Graphical presentation of Tm values for23IL0064 as a function of NaCl concentration (FIG. 32(A)) and ofmannitol concentration (FIG. 32(B)). Tm's were obtained in the thermalshift assay at 0.1 mg/mL.

FIG. 33. Graphical representation of the melting temperatures measuredfor 23IL0075 as a function of the pH, in 10 mM acetate, in 10 mMhistidine, and in 10 mM phosphate buffer. The protein concentration was0.2 mg/mL. Scanning was performed at 1° C./min, starting at 30° C.

FIG. 34. SE-HPLC chromatograms (OD280) of 23IL0064 in 40 mM histidine(pH 6.0)/50 mM NaCl. The start sample was compared to samples afterconcentration. No aggregate formation due to concentration of the samplein the histidine buffer was observed. The SE-HPLC was run with aPhenomenex BIOSEP SEC S-2000 column, with D-PBS as mobile phase at 0.2mL/min.

FIG. 35. Log-values of the soluble protein concentration (Y-axis) vs.PEG6000 concentration (%, X-axis). The right upper panel represents anexample of how the linear regression analysis is performed on theobtained data points to determine the intercept with the X-axis (fromwhich the theoretical solubility at zero % PEG6000 can be deduced). Inthe histidine buffer, the experiment was performed at two concentrationswhich are presented in the two bottom graphs. For these two graphs noregression was possible.

FIG. 36. Log-values of the soluble protein concentration (Y-axis) ofp23IL0064 and 23IL0075 in 10 mM phosphate buffer pH 7 with 50 mM NaClvs. PEG6000 concentration (X-axis). By regression analysis andextrapolation to a zero concentration of PEG6000, the theoreticalmaximum protein concentrations (apparent solubility values) werecalculated and were for both proteins approximately 50 mg/mL. Thisnumber should only be used after confirmation with other techniques.

FIG. 37. Log-values of the soluble protein concentration (Y-axis) ofp23IL0064 and p23IL0075 in 40 mM histidine pH 6.0 buffer with 50 mM NaClvs. PEG6000 concentration (X-axis). In this graph regression was notpossible since no precipitation occurred in the PEG % window explored.

FIG. 38. SE-HPLC chromatograms (280 nm) of 25 μg 23IL0064 non-stressedand 37° C. (3w and 4w)-stressed samples in D-PBS. SE-HPLC was run onTSK-GEL G2000SWXL with D-PBS.

FIG. 39. RP-HPLC chromatograms (280 nm) of 25 μg 23IL0064 non-stressedand 37° C. (4w)-stressed sample in D-PBS. RP-HPLC was run on ZORBAX C-3column with a water/acetonitrile 0.1% TFA gradient.

FIG. 40. Graphical representation of the % peak area of the differentRP-HPLC peaks of 23IL0064 and product related substances after 6 weeksstress at 37° C. in different formulation buffers. The first barrepresents the reference sample, stored at −80° C. until analysis. Thepre-peak 2 was present in the start sample and did not increase duringthe storage at 37° C.

FIG. 41. Overlay of RP-HPLC chromatograms (280 nm) of 23IL0064 in 20 mMHistidine pH 6.5 (conc. 22.4 mg/mL). Comparison of 6 weeks 37° C., 6weeks 25° C., 6 weeks 4° C., and −80° C. reference. RP-HPLC was run onZORBAX 300SB-C3 column with a water/acetonitrile 0.1% TFA gradient0.3%/min) at 75° C.

FIG. 42. SDS-PAGE of stability samples of 23IL0064 in 20 mM Histidine pH6.5 CONC stressed for 6 weeks at 4° C., 25° C., 37° C. and its −80° C.Reference.

FIG. 43. Elastic light scattering as measured at 500 nm as a function ofthe temperature for 23IL0075 samples with different concentrations (asindicated in the legend).

FIG. 44. Elastic light scattering as measured at 500 nm as a function ofthe temperature for 250 μg/mL 23IL0075 in 10 mM phosphate pH 6.0. Thetemperature of the onset of aggregate formation is determined by meansof linear fit.

FIG. 45. Elastic light scattering as measured at 500 nm as a function ofthe temperature for 250 μg/mL 23IL0075 in 10 mM Acetate pH 6.0. Thetemperature of the onset of aggregate formation is determined by meansof linear fit.

FIG. 46. Elastic light scattering as measured at 500 nm as a function ofthe temperature for 2501 g/mL 23IL0075 in 10 mM Histidine pH 6.0. Thetemperature of the onset of aggregate formation is determined by meansof linear fit.

FIG. 47(A), FIG. 47(B). Graphical representation of the opalescence(measured by OD500) (FIG. 47(A)) and the % oligomers (% pre-peak)detected in SE-HPLC (FIG. 47(B)) after freeze/thaw stress. Thepercentage of oligomers in the reference sample was approximately 0.4%.In this study, an acetate, a histidine and a phosphate buffer werecompared, in combination with mannitol or a mixture of mannitol andglycine as excipients, and Tween 80 or poloxamer in two differentconcentrations as surfactants.

FIG. 48(A), FIG. 48(B). Graphical representation of the opalescence(measured by OD500) (FIG. 48(A)) and the % oligomers detected in SE-HPLC(FIG. 48(B)) after shear stress. The percentage of oligomers in thereference sample was approximately 0.4%. In this study, an acetate, ahistidine and a phosphate buffer were compared, in combination withmannitol or a mixture of mannitol and glycine as excipients, and Tween80 or poloxamer in two different concentrations as surfactants.

FIG. 49(A), FIG. 49(B). Graphical representation of the opalescence(measured by OD500) (FIG. 49(A)), and the % oligomers detected inSE-HPLC (FIG. 49(B)) in different histidine buffers after freeze/thawstress. The percentage of oligomers in the reference sample wasapproximately 0.4%. *For one sample the condition without detergent wasincluded.

FIG. 50(A), FIG. 50(B) and FIG. 50(C). Graphical representation of theopalescence (measured by OD500) (FIG. 50(A)), the % oligomers detectedin SE-HPLC (FIG. 50(B)), and the % activity (albumin binding) of theNanobody measured on Biacore (FIG. 50C) after shear stress. Thepercentage of oligomers in the reference sample was approximately 0.4%.In this study no detergents were included, to mimic the situation duringthe final concentration step of the DSP process.

FIG. 51. SE-HPLC chromatograms (280 nm) of 23IL0075 at 25 mg/mL in 10 mMHistidine pH 6.0, 10% sucrose, 0.005% TWEEN (polysorbate) 80: thereference sample compared to 6 weeks storage at 25° C. and 37° C.

FIG. 52: Hallmark Residues in single variable domains.

FIG. 53a and FIG. 53b : Illustration of various non-fused dimers (i.e.NFDs) and comparison with the conventional genetically fused molecules.Single Variable Domains in each construct or NFD may be different (FIG.53a +FIG. 53b ) or identical (FIG. 53a ). The dashed line is a schematicinteraction between the 2 VH domains that confer the NFD its stability(indicated here are surface interactions but these can also be otherinteraction as described in the invention herein).

FIG. 54: Protein A affinity purification of polypeptide A (SEQ ID NO: 7)under conditions resulting in significant amounts of NFDs. The proteinwas loaded on a small column (40011 resin MabSelectXtra, GE Healthcare)and eluted via injection of glycine [100 mM, pH=2.5]. The pH of theeluted Nanobody® solution was immediately neutralized using 1M Tris pH8.8.

FIG. 55: Size exclusion chromatography of Protein A affinity purifiedpolypeptide A. Separation of concentrated polypeptide A (fraction 6, seeFIG. 54) on an analytical SUPERDEX 75 column (GE Healthcare). TheNanobody® fraction was resolved into two specific fractionscorresponding to the molecular weight of monomeric and dimericpolypeptide A (position of molecular weight markers is indicated).Analysis via SDS-PAGE (right panel) did not reveal any differencebetween the two, indicating that under native conditions they behave asmonomer and dimer. The latter is converted into a monomer conformationupon denaturation (SDS detergent and heat treatment).

FIG. 56: Protein A affinity purification of polypeptide A at low columnload. A limited amount of protein [approx. 2.5 mg/ml resin] was loadedon a small column (4001 resin MabSelectXtra, GE Healthcare) and elutedvia injection of glycine [100 mM, pH=2.5]. The pH of the elutedNanobody® solution was immediately neutralized using 1M Tris pH 8.8.

FIG. 57: Size exclusion chromatography of Protein A affinity purifiedpolypeptide A. Separation of concentrated polypeptide A (fraction 7, seeFIG. 56) on an analytical SUPERDEX 75 column (GE Healthcare). TheNanobody® fraction was resolved into a specific fraction correspondingto the molecular weight of monomeric polypeptide.

FIG. 58: Protein A elution of Polypeptide A. The pretreated periplasmicextract was loaded on a Protein A MabSelectXtra column, followed by aPBS wash until stable baseline. Elution was carried out via a pH shiftusing 100 mM glycine pH=2.5 (dotted line).

FIG. 59: Size Exclusion Chromatography of Polypeptide A monomer anddimer. The pre-peak (fraction 2) contains the dimeric Polypeptide Awhich was used in the stability studies.

FIG. 60: Size exclusion chromatography of heat treated samples ofdimeric Polypeptide A. Polypeptide A NFD (at 0.68 mg/ml) was used inseveral experiments: 20 μl dimer fractions were diluted with 90 μl D-PBSand incubated at different temperatures and 100 μl was analysed on aSUPERDEX 75™ 10/300GL column equilibrated in D-PBS.

FIG. 61: Size exclusion chromatography of pH treated samples ofPolypeptide A NFD. Polypeptide A NFD (at 0.68 mg/ml) was used in severalexperiments: 20 μl dimer samples were diluted with 90 μl [100 mMPiperazine pH=10.2] or 90μl [100 mM Glycine, pH=2.5] and incubatedovernight (ON) at 4° C. The control was incubated in D-PBS. Samples wereanalysed via SEC the next day. The incubation at elevated pH had noeffect on the dissociation whereas low pH (glycine pH=2.5) resulted inapprox 15% monomer. A more drastic incubation in 1% TFA during 15 min atroom temperature resulted in almost 100% monomer.

FIG. 62: Size exclusion chromatography of combined heat/organic solventtreated samples of Polypeptide A NFD. Polypeptide A NFD (at 0.68 mg/ml)was used in several experiments: 20 μl dimer fractions were diluted with90 μl [10% Isopropanol] or 90 μl [30% Isopropanol] and incubatedovernight (ON) at 4° C. or 15 minutes at 20° C. Combined treatments(heat and Isopropanol) were carried out during 15 minutes. The controlwas incubated in D-PBS. Samples were analysed via SEC. The incubation atelevated temperature with organic solvent resulted in accelerateddissociation into monomer.

FIG. 63: Size exclusion chromatography of ligand-NFD complex formation:20 μl samples of Ligand A (SEQ ID NO:13) was diluted in 90 μl [HBS-EP(BIACORE)+0.5M NaCl] and incubated for several hours at RT (ligand mix).Then NFD or Polypeptide A was added and after a short incubation(typically 30 min) the material was resolved via SEC. Polypeptide A[3.91 mg/ml]: 17 μl [ 1/10 diluted in HBS-EP] was added to the ligandmix and 100 μl was injected.

FIG. 64: The molecular weight (MW) of polypeptide A, Ligand A,Polypeptide A+Ligand A, NFD-Di of Polypeptide A, and NFD-Di ofPolypeptide A+Ligand A was calculated (see Table 46 for read out fromthis figure) based on curve fitting of Molecular weight standards(Biorad #151-1901) run on the same column under same conditions.

FIG. 65: Monomer of Polypeptide B as present in the dimer (top) and anisolated monomer of polypeptide B (bottom).

FIG. 66: Polypeptide B-dimer (an example of a NFD-Mo). Framework 4 ofmonomer A is replaced by framework 4 of monomer B and vice versa.

FIG. 67: Electron-density of monomer B in black. Monomer A is shown ingrey ribbon.

FIG. 68: Polypeptide B (top) and polypeptide F with Pro at position 45(bottom).

FIG. 69: Size exclusion chromatography of Polypeptide B material elutedfrom Protein A affinity column on SUPERDEX 75 XK 26/60 column.

FIG. 70: Fluorescence emission SYPRO orange in the presence ofpolypeptide B and polypeptide B-dimer.

FIG. 71: Unfolding of Polypeptide B monomer and Polypeptide B-dimer infunction of Guanidinium Hydrochloride concentration. Unfolding wasmonitored by intrinsic fluorescence measurements and thereby usingcenter of spectral mass (CSM) as unfolding parameter.

FIG. 72: Purity was analysed on a Coomassie stained gel (Panel A:Polypeptide G; Panel B: Polypeptide H).

FIG. 73: Binding of polypeptide F, G, and H on HSA.

FIG. 74: The 280 nm SE-HPLC chromatograms of Polypeptide I formulated inphosphate buffer (2 weeks storage) with either 50 mM NaCl, 100 mM NaClor 10% mannitol. A zoom on the main peak is shown as inset.

FIG. 75(A), FIG. 75(B): Figure demonstrating the time-dependent decrease(FIG. 75(A)) and increase (FIG. 75(B)) of the surface area of,respectively, the main peak and % dimers observed in SE-HPLC analysis ofPolypeptide I formulated in different buffers and stored for 10 weeks at37° C.

FIGS. 76-79: Overview of the results obtained for thermal stabilitytesting of Polypepetides J and K.

FIG. 80: Overview of the results obtained in thermal stability testingof Polypeptide J in Tris buffer pH 7.2 or Histidine pH 6.5, withsucrose, glycine or mannitol added as excipient.

FIG. 81: Overlay of the SE-HPLC chromatograms of IL6R304 formulated at10 mg/mL stored for 3 weeks at 37° C. Inset, zoom on the main peak todemonstrate the buffer-dependent differences in % aggregates.

FIG. 82: Figure demonstrating the buffer-dependent differences in %aggregates (peak surface area in SE-HPLC) that were observed in thestability samples of Polypeptide J and Polypeptide K stored for 1 weekat 37° C.

FIG. 83: Figure demonstrating the time-dependent increase of the %oligomers/aggregates (Y-axis) observed in SE-HPLC analysis ofPolypeptide J stored for up to 5 weeks at 37° C. (A) in the buffersindicated in the graph. The % oligomers/aggregates is expressed as thesum of the % peak surface areas of prepeak 1a, prepeak 1b and prepeak 2relative to the total peak surface area.

FIG. 84: Time-dependent and buffer-dependent increase in the % oligomersobserved in the stability samples stored for up to 5 weeks at 37° C., ata concentration of 10 mg/mL in the buffers indicated in the graph.

FIG. 85(A), FIG. 85(B) and FIG. 85(C): Overlay of the SE-HPLCchromatograms from Polypeptide J after storage for up to 8 weeks at +37°C. in 10 different formulation buffers. A zoom on the main peak (inset)demonstrates the time-dependent increase of the surface area of prepeaksand postpeak. FIG. 85(A): storage for 1 week. FIG. 85(B): storage for 4weeks. FIG. 85(C): storage for 8 weeks.

FIG. 86: SE-HPLC analysis of Polypeptide J samples stored for 8 weeks at37° C. in L-histidine buffer (buffers 1-5) compared to phosphate buffer(buffers 6-10). The amount of oligomers was lowest in buffer 3.

FIG. 87: Kinetics of oligomer formation upon storage of Polypeptide J inthe different buffers.

DETAILED DESCRIPTION

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10th Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein.

As used herein, the term “isolated” in the context of a polypeptiderefers to a polypeptide which is substantially free of cellular materialor contaminating proteins from the cell or tissue source from which itis derived, or substantially free of chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of a polypeptide in whichthe polypeptide is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, a polypeptide thatis substantially free of cellular material includes preparations of apolypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein, polypeptide, peptide, or antibody (alsoreferred to as a “contaminating protein”). When the polypeptide isrecombinantly produced, it may also be substantially free of culturemedium, i.e., culture medium represents less than about 20%, 10%, or 5%of the volume of the polypeptide preparation. When the polypeptide isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the polypeptide. Accordingly, such preparations of apolypeptide have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.In a specific embodiment, an “isolated” polypeptide is purified by amulti-step purification process that comprises two chromatography steps(e.g. cation exchange and anion exchange), a 100K ultrafiltration step,followed by a buffer exchange and concentration step inUltrafiltration/Diafiltration mode.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., amonkey, such as a cynomolgus monkey, chimpanzee, baboon and a human),and more preferably a human. In a certain embodiment, the subject is amammal, preferably a human, with one or more diseases or disorders. Inanother embodiment, the subject is a mammal, preferably a human, at riskof developing one or more diseases and/or disorders.

The terms “stability” and “stable” as used herein in the context of aformulation comprising a polypeptide comprising one or more singlevariable domains refer to the resistance of the polypeptide in theformulation to aggregation, to the formation of degradation productsand/or to the formation of fragmentation products under giventransportation and/or storage conditions. Apart from this and/or inaddition, the “stable” formulations of the invention retain biologicalactivity under given transportation and/or storage conditions. Thestability of said polypeptide can be assessed by degrees of aggregation,degradation and/or fragmentation (as measured e.g. by SE-HPLC, RP-HPLC,IEX-HPLC, subvisible particle counting, analytical ultracentrifugation,dynamic light scattering, OD320/OD280 ratio measurement, elastic lightscattering, etc.), and/or by % of biological activity (as measured e.g.by ELISA, BIACORE, etc.) compared to a reference formulation. Forexample, a reference formulation may be a reference standard frozen at−20° C. or below −65° C. (such as e.g. −80° C.) consisting of the samepolypeptide at the same concentration in D-PBS or consisting of the samepolypeptide at the same concentration and in the same buffer as thestressed samples but without applying the stress conditions, whichreference formulation regularly gives a single peak by SE-HPLC, RP-HPLCand/or IEX-HPLC and/or keeps its biological activity in BIACORE and/orELISA.

“Solubility” is often described as the maximum achievable proteinconcentration whereby all of the protein remains in solution. At thisconcentration the protein should still be monomeric and free ofaggregates. For determining protein solubility only a limited number of(mostly empirical) techniques are currently available. A first andpopular technique consists of concentrating the sample by usingcentrifugal ultrafiltration up to the point where an opalescent solutionis formed. Subsequently, the insoluble fraction is removed and theprotein content of the supernatant is measured. Centrifugalconcentrating devices such as for example VIVASPIN concentrators with amolecular weight cut-off of 5 kDa can be used but require reasonableamounts of protein. Solubility can also be monitored using an inertmacromolecule such as polyethylene glycol (PEG; Mr>6,000), whichprecipitates proteins primarily through an excluded volume effect, aprocess that can be generally understood in terms of a simple colloidalphase separation. A logarithmic linear relationship between proteinsolubility and weight percent polyethylene glycol can be obtained, andfrom this plot the intercept yields the solubility value. The term “goodsolubility” of the polypeptide of the invention, as used herein, meansthat no or little precipitation is observed of the polypeptide of theinvention during downstream processing (DSP) and/or during storage for ashort or longer time at 5° or −20° C. at concentrations ranging from20-200 mg/mL or more. The formation of precipitates (oligomers or otherparticulates) can be measured e.g. by SE-HPLC, OD320/OD280 ratiomeasurement and/or elastic light scattering. Preferably, thepolypeptides present in the formulations of the present invention have asolubility of at least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL,at least 50 mg/mL, at least 60 mg/mL, at least 65 mg/mL, at least 70mg/mL, at least 80 mg/mL, at least 90 mg/mL, at least 100 mg/mL, atleast 110 mg/mL, at least 120 mg/mL, at least 130 mg/mL, at least 140mg/mL, at least 150 mg/mL, at least 200 mg/mL or even more. Preferably,the OD320/OD280 ratio of the formulations of the present invention is0.05 or lower, such as 0.01 or lower or 0.005 or lower. The scatteringin the formulation of the present invention should be within detectionlimit and preferably lower than 1000 abs, such as 750 abs or lower or500 abs or lower.

The phrase “low to undetectable levels of aggregation” as used hereinrefers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1% or no more than 0.5%aggregation by weight of protein. Unless explicitly referred todifferently, aggregation as used in the present invention means thedevelopment of high molecular weight aggregates, i.e. aggregates with anapparent molecular weight of more/higher than the apparent molecularweight observed in SE-HPLC analysis for dimers of the polypeptide of theinvention (such as e.g. 44 kDa as observed for SEQ ID NO: 4; 36-38 kDaas observed for SEQ ID NO's 1-3; and 36 kDa as observed for SEQ ID NO: 5in SE-HPLC) in comparison with molecular weight markers. Aggregation canbe assessed by various methods known in the art. Without being limiting,examples include high performance size exclusion chromatography(SE-HPLC), subvisible particle counting, analytical ultracentrifugation(AUC), dynamic light scattering (DLS), static light scattering (SLS),elastic light scattering, OD320/OD280 measurement, Fourier TransformInfrared Spectroscopy (FTIR), circular dichroism (CD), urea-inducedprotein unfolding techniques, intrinsic tryptophan fluorescence and/ordifferential scanning calorimetry techniques.

The term “low to undetectable levels of fragmentation and/ordegradation” as used herein refers to samples containing equal to ormore than 80%, 85%, 90%, 95%, 98% or 99% of the total protein, forexample, in a single peak as determined by SE-HPLC, RP-HPLC and/orIEX-HPLC, representing the non-degraded polypeptide, and containing noother single peaks having more than 5%, more than 4%, more than 3%, morethan 2%, more than 1%, or more than 0.5% of the total protein in each.

The term “very little to no loss of the biological activities” as usedherein refers to single variable domain activities, including but notlimited to, specific binding abilities of the single variable domain tothe target of interest as measured by various immunological assays,including, but not limited to ELISAs and/or by Surface Plasmon Resonance(BIACORE). In one embodiment, the single variable domains of theformulations of the invention retain at least 50%, preferably at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% or more ofthe ability to specifically bind to an antigen as compared to areference formulation, as measured by an immunological assay known toone of skill in the art or described herein. For example, an ELISA basedassay (e.g. as described in the Example section) may be used to comparethe ability of the single variable domain to specifically bind to itstarget. A “reference formulation” as used herein refers to a formulationthat is frozen at a temperature of −20±5° C. or at below −64° C. (suchas e.g. at −80° C.) consisting of the same single variable domain at thesame concentration in D-PBS or consisting of the same single variabledomains at the same concentration in the same buffer/excipients as thestressed samples but without applying the stress conditions, whichreference formulation regularly gives a single peak by SE-HPLC, RP-HPLCand/or IEX-HPLC and/or keeps its biological activity in BIACORE and/orELISA.

The phrase “pharmaceutically acceptable” as used herein means approvedby a regulatory agency of the Federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopoeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. In this sense, it should be compatible with the otheringredients of the formulation and not eliciting an unacceptabledeleterious effect in the subject.

According to the European Pharmacopoeia, a solution is consideredisotonic if it has an osmolality of 290±30 mOsm/kg. Osmolalitymeasurements were therefore performed on the different formulations usedin the stability studies. Isotonicity can be measured by, for example, avapor pressure or ice-freezing type osmometer.

As used herein, the term “effective amount” refers to the amount of anagent (e.g. a prophylactic or therapeutic agent) which is sufficient toreduce and/or ameliorate the severity and/or duration of one or morediseases and/or disorders.

The term “polyol” as used herein refers to sugars that contains manyhydroxyl (—OH) groups compared to a normal saccharide. Polyols includealcohols and carbohydrates such as mannitol, sorbitol, maltitol,xylitol, isomalt, erythritol, lactitol, sucrose, glucose, galactose,fructose, fucose, ribose, lactose, maltose and cellubiose.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the prevention, treatmentand/or management of one ore more diseases and/or disorders. In thecontext of the present invention, the term “therapeutic agent” refers toa polypeptide comprising one or more single variable domains. In certainother embodiments, the term “therapeutic agent” refers to an agent otherthan the polypeptide of the invention which might be used in theformulation.

As used herein, the term “therapeutically effective amount” refers tothe amount of a therapeutic agent (e.g. a polypeptide comprising one ormore single variable domains), that is sufficient to reduce the severityof one or more diseases and/or disorders.

The term “excipient” as used herein refers to an inert substance whichis commonly used as a diluent, vehicle, preservative, binder orstabilizing agent for drugs which imparts a beneficial physical propertyto a formulation, such as increased protein stability, increased proteinsolubility, and/or decreased viscosity. Examples of excipients include,but are not limited to, proteins (e.g., serum albumin), amino acids(e.g., aspartic acid, glutamic acid, lysine, arginine, glycine),surfactants (e.g., SDS, TWEEN (polysorbate) 20, TWEEN (polysorbate) 80,poloxamers, polysorbate and nonionic surfactants), saccharides (e.g.,glucose, sucrose, maltose and trehalose), polyols (e.g., mannitol andsorbitol), fatty acids and phospholipids (e.g., alkyl sulfonates andcaprylate). For additional information regarding excipients, seeRemington's Pharmaceutical Sciences (by Joseph P. Remington, 18th ed.,Mack Publishing Co., Easton, Pa.), which is incorporated herein in itsentirety.

The term “variable domain” or “immunoglobulin variable domain” refers tothe part or domain of an immunoglobulin molecule or antibody which ispartially or fully responsible for antigen binding. The term “singlevariable domain” or “immunoglobulin single variable domain” (both termsare used interchangeably), defines molecules wherein the antigen bindingsite is present on, and formed by, a single immunoglobulin domain. Thissets single variable domains apart from “conventional” immunoglobulinsor their fragments, wherein two immunoglobulin domains, in particulartwo “variable domains” interact to form an antigen binding site.Typically, in conventional immunoglobulins, a heavy chain variabledomain (VH) and a light chain variable domain (VL) interact to form anantigen binding site. In this case, the complementarity determiningregions (CDRs) of both VH and VL will contribute to the antigen bindingsite, i.e. a total of 6 CDRs will be involved in antigen binding siteformation.

In contrast, the binding site of a single variable domain is formed by asingle VH or VL domain. Hence, the antigen binding site of a singlevariable domain is formed by no more than three CDRs. The term “singlevariable domain” does comprise fragments of conventional immunoglobulinswherein the antigen binding site is formed by a single variable domain.

Generally, single variable domains will be amino acid sequences thatessentially consist of 4 framework regions (FR1 to FR4 respectively) and3 complementarity determining regions (CDR1 to CDR3 respectively); orany suitable fragment of such an amino acid sequence (which will thenusually contain at least some of the amino acid residues that form atleast one of the CDR's). Such single variable domains and fragments aremost preferably such that they comprise an immunoglobulin fold or arecapable for forming, under suitable conditions, an immunoglobulin fold.As such, the single variable domain may for example comprise a lightchain variable domain sequence (e.g. a V_(L) sequence) or a suitablefragment thereof; or a heavy chain variable domain sequence (e.g. aV_(H) sequence or V_(HH) sequence) or a suitable fragment thereof; aslong as it is capable of forming a single antigen binding unit (i.e. afunctional antigen binding unit that essentially consists of the singlevariable domain, such that the single antigen binding domain does notneed to interact with another variable domain to form a functionalantigen binding unit, as is for example the case for the variabledomains that are present in for example conventional antibodies and scFvfragments that need to interact with another variable domain—e.g.through a V_(H)/V_(L) interaction—to form a functional antigen bindingdomain).

In one aspect of the invention, the single variable domains are lightchain variable domain sequences (e.g. a V_(L) sequence), or heavy chainvariable domain sequences (e.g. a V_(H) sequence); more specifically,the single variable domains can be heavy chain variable domain sequencesthat are derived from a conventional four-chain antibody or heavy chainvariable domain sequences that are derived from a heavy chain antibody.

The single variable domain may be a domain antibody (or an amino acidsequence that is suitable for use as a domain antibody), a single domainantibody (or an amino acid sequence that is suitable for use as a singledomain antibody), a “dAb” (or an amino acid sequence that is suitablefor use as a dAb) or a Nanobody® (as defined herein, and including butnot limited to a V_(HH) sequence) [Note: Nanobody® and Nanobodies® areregistered trademarks of Ablynx N.V.]; other single variable domains, orany suitable fragment of any one thereof. For a general description of(single) domain antibodies, reference is also made to the prior artcited herein, as well as to EP 0 368 684. For the term “dAb's”,reference is for example made to Ward et al. 1989 (Nature 341 (6242):544-546), to Holt et al. 2003 (Trends Biotechnol. 21(11): 484-490); aswell as to for example WO 04/068820, WO 06/030220, WO 06/003388 andother published patent applications of Domantis Ltd. It should also benoted that, although less preferred in the context of the presentinvention because they are not of mammalian origin, single variabledomains can be derived from certain species of shark (for example, theso-called “IgNAR domains”, see for example WO 05/18629).

In particular, the polypeptides of the invention may comprise one ormore Nanobodies or a suitable fragment thereof. For a furtherdescription of V_(HH)'s and Nanobodies, reference is made to the reviewarticle by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302); as well as to the following patent applications, which arementioned as general background art: WO 94/04678, WO 95/04079 and WO96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1 134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694,WO 03/054016 and WO 03/055527 of the Vlaams Instituut voorBiotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.;WO 01/90190 by the National Research Council of Canada; WO 03/025020(=EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867,WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, byAblynx N.V. and the further published patent applications by Ablynx N.V.Reference is also made to the further prior art mentioned in theseapplications, and in particular to the list of references mentioned onpages 41-43 of the International application WO 06/040153, which listand references are incorporated herein by reference. As described inthese references, Nanobodies (in particular V_(HH) sequences andpartially humanized Nanobodies) can in particular be characterized bythe presence of one or more “Hallmark residues” in one or more of theframework sequences. A further description of the Nanobodies, includinghumanization and/or camelization of Nanobodies, as well as othermodifications, parts or fragments, derivatives or “Nanobody fusions”,multivalent constructs (including some non-limiting examples of linkersequences) and different modifications to increase the half-life of theNanobodies and their preparations can be found e.g. in WO 08/101985 andWO 08/142164.

The total number of amino acid residues in a Nanobody can be in theregion of 110-120, is preferably 112-115, and is most preferably 113. Itshould however be noted that parts, fragments, analogs or derivatives(as further described herein) of a Nanobody are not particularly limitedas to their length and/or size, as long as such parts, fragments,analogs or derivatives meet the further requirements outlined herein andare also preferably suitable for the purposes described herein.

Thus, in the meaning of the present invention, the term “single variabledomain” comprises polypeptides which are derived from a non-humansource, preferably a camelid, preferably a camelid heavy chain antibody.They may be humanized, as previously described. Moreover, the termcomprises polypeptides derived from non-camelid sources, e.g. mouse orhuman, which have been “camelized”, as previously described.

The term “single variable domain” also encompasses variable domains ofdifferent origin, comprising mouse, rat, rabbit, donkey, human andcamelid variable domains; as well as fully human, humanized or chimericvariable domains. For example, the invention comprises camelid variabledomains and humanized camelid variable domains, or camelized variabledomains, e.g. camelized dAb as described by Ward et al (see for exampleWO 94/04678 and Davies and Riechmann (1994, FEBS Lett. 339(3): 285-290)and (1996, Protein Eng. 9(6): 531-537)). Moreover, the inventioncomprises fused variable domains, e.g. multivalent and/or multispecificconstructs (for multivalent and multispecific polypeptides containingone or more V_(HH) domains and their preparation, reference is also madeto Conrath et al. 2001 (J. Biol. Chem. 276: 7346-7350) as well as to forexample WO 96/34103 and WO 99/23221).

Unless indicated otherwise, the term “immunoglobulin sequence”—whetherused herein to refer to a heavy chain antibody or to a conventional4-chain antibody—is used as a general term to include both the full-sizeantibody, the individual chains thereof, as well as all parts, domainsor fragments thereof (including but not limited to antigen-bindingdomains or fragments such as V_(HH) domains or V_(H)/V_(L) domains,respectively). The terms antigen-binding molecules or antigen-bindingprotein are used interchangeably with immunoglobulin sequence, andinclude Nanobodies.

The single variable domains provided by the invention are preferably inessentially isolated form (as defined herein), or form part of apolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more single variable domains and which mayoptionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more single variable domains may beused as a binding unit in such a polypeptide, which may optionallycontain one or more further amino acid sequences that can serve as abinding unit (i.e. against one or more other targets), so as to providea monovalent, multivalent or multispecific polypeptide of the invention,respectively as e.g. described in WO 08/101985, WO 08/142164, WO09/068625, WO 09/068627 and WO 08/020079. Such a protein or polypeptidemay also be in essentially isolated form (as defined herein) and themethods of the present invention for the expression and/or production ofsingle variable domains equally apply to polypeptides comprising one ormore single variable domains.

According to the invention, the term “single variable domain” maycomprise constructs comprising two or more antigen binding units in theform of single variable domain, as outlined above. For example, two (ormore) variable domains with the same or different antigen specificitycan be linked to form e.g. a bivalent, trivalent or multivalentconstruct. By combining variable domains of two or more specificities,bispecific, trispecific etc. constructs can be formed. For example, avariable domain according to the invention may comprise two variabledomains directed against target A, and one variable domain againsttarget B. Such constructs and modifications thereof, which the skilledperson can readily envisage, are all encompassed by the term variabledomain as used herein and are also referred to as “polypeptide of theinvention” or “polypeptides of the invention”.

As further described in paragraph m) on page 53 of WO 08/020079, anamino acid sequence (such as a Nanobody, an antibody, a polypeptide ofthe invention, or generally an antigen binding protein or polypeptide ora fragment thereof) that can (specifically) bind to, that has affinityfor and/or that has specificity for a specific antigenic determinant,epitope, antigen or protein (or for at least one part, fragment orepitope thereof) is said to be “against” or “directed against” saidantigenic determinant, epitope, antigen or protein.

The polypeptide comprising one or more single variable domains for usein the formulation of the invention may be therapeutic or prophylactic,and may be useful in the treatment and/or management of one or morediseases. In one specific aspect, the polypeptide has at least twosingle variable domains. In another specific aspect, the polypeptide hasat least three single variable domains. Preferably, the polypeptidecomprises at least one single variable domain directed against HSA. Inanother specific aspect, the polypeptide comprises at least a singlevariable domain against RANKL. In another specific aspect, thepolypeptide comprises at least a single variable domain against IL-6R.In another specific aspect, the polypeptide comprises at least a singlevariable domain against IL-23. More preferably, the polypeptide isdirected against and/or specifically binds RANKL and HSA, IL-6R and HSAand/or IL-23 and HSA. In yet another aspect, polypeptide comprises atleast a single variable domain against RANKL and at least a singlevariable domain against HSA. In yet another aspect, polypeptidecomprises at least a single variable domain against IL-6R and at least asingle variable domain against HSA. In yet another aspect, polypeptidecomprises at least a single variable domain against IL-23 and at least asingle variable domain against HSA. In yet another aspect, polypeptidecomprises at least two single variable domains against RANKL and atleast a single variable domain against HSA. In yet another aspect,polypeptide comprises at least two single variable domains against IL-6Rand at least a single variable domain against HSA. In yet anotheraspect, polypeptide comprises at least two single variable domainsagainst IL-23 and at least a single variable domain against HSA. In apreferred aspect, the single variable domains used in the polypeptide ofthe invention are selected from WO 08/142164 (such as e.g. SEQ ID NO's:745 and/or 791 of WO 08/142164), WO 08/020079, WO 09/068627 (such ase.g. SEQ ID NO's 2578, 2584 and/or 2585 of WO 09/068627), PCTapplication No. PCT/EP2010/054747 by Ablynx N.V., PCT application No.PCT/EP2010/054764 by Ablynx N.V. (such as e.g. SEQ ID NO's: 66 and/or 98of PCT/EP2010/054764) and WO 08/028977 (such as e.g. SEQ ID NO: 62 of WO08/028977). Preferred polypeptides of the invention are selected fromSEQ ID NO's: 1 to 6.

The concentration of polypeptide of the invention present in theformulation can by any concentration of the polypeptide that providesthe desired effect to the subject. In a preferred aspect, theconcentration of the polypeptide of the invention is from 1 to 200 mg/mLsuch as about 1 mg/mL, about 2 mg/mL, about 5 mg/mL, about 10 mg/mL,about 15 mg/mL or about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about50 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 80mg/mL, about 90 mg/mL or about 100 mg/mL or more. In certainembodiments, the concentration of polypeptide of the invention can be110 mg/mL or more, 120 mg/mL or more, 130 mg/mL or more, 140 mg/mL ormore, 150 mg/mL or more or even 200 mg/mL or more. In a specific aspect,a formulation of the invention comprises about 10 mg/mL of polypeptideof the invention.

The formulation of the invention comprises an aqueous carrier having apH of 5.5 to 8.0 and a polypeptide as defined above (“polypeptide of theinvention”) comprising one or more single variable domains at aconcentration of 1 mg/mL to 200 mg/mL, said formulation being formulatedfor administration to a human subject, wherein said formulation furthercomprises one or more components selected from:

-   -   a) A buffer at a concentration of 10 mM to 100 mM selected from        the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0,        MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;    -   b) An excipient at a concentration of 1% to 20%;    -   c) A surfactant at a concentration of 0.001% to 1% selected from        TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer;        and        wherein said formulation has an inorganic salt concentration of        150 mM or lower.

The stable formulations of the present invention comprise polypeptidesof the invention that have a good solubility and a high stability evenduring transportation and/or long periods of storage and that exhibitlittle to no aggregation. In addition to the polypeptide of theinvention, the formulations of the present invention comprise at leastan aqueous carrier and a buffer. The carrier used in the formulation ofthe invention should be a liquid carrier. Preferably the carrier is anaqueous carrier such as e.g. distilled water, MILLI-Q water or Water forInjection (WFI).

The formulation should not contain inorganic salt at a concentration ofmore than 150 mM. Without being limiting, inorganic salts for use in theformulation of the invention can be selected from NaCl and KCl.Accordingly the formulation of the invention has an inorganic saltconcentration of 150 mM or lower, preferably 120 mM or lower, or 100 mMor lower, more preferably 90 mM or lower, 80 mM or lower, 75 mM orlower, such as 50 mM or lower or even 40 mM or lower, 25 mM or lower, 10mM or lower or 5 mM or lower. Most preferably, the formulation does notcontain any inorganic salt.

The pH of the formulation of the invention generally should not be equalto the isoelectric point of the particular polypeptide of the inventionpresent in the formulation and may range from about 5.5 to about 8.0, orfrom about 6.0 to about 7.5, preferably from about 6.2 to 7.5, fromabout 6.5 to 7.5, most preferably from about 6.5 to 7.0. In a specificaspect, the formulation of the invention has a pH of about 6.5. Inanother specific aspect, the formulation of the invention has a pH ofabout 7.0. In yet another specific aspect, the formulation of theinvention has a pH of about 6.0.

The formulation may be buffered by a buffer selected from the groupconsisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,succinate pH 6.0-6.5 and acetate pH 5.5-6.0, preferably hepes pH 7.0 orhistidine pH 6.0-6.5, such as histidine pH 6.5 or histidine pH 6.0.

The concentration of the buffer present in the formulation of theinvention may range from 1 mM to 100 mM, 5 mM to 100 mM, 5 mM to 75 mM,5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25 mM, 10 mM to 20 mM. In aspecific aspect, the concentration of buffer in the formulations of theinvention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM,or 100 mM. Preferably, the concentration is between 10 and 20 mM, suchas 10 mM or 15 mM.

Any form of histidine suitable for formulation and parenteraladministration may be used in the formulation of the invention. Thepurity of histidine should be at least 98%, at least 99%, or at least99.5%. In a specific aspect, a formulation of the invention comprises 15mM histidine buffer pH 6.5. In another specific aspect, a formulation ofthe invention comprises 10 mM histidine buffer pH 6.0.

Apart from or in addition to histidine, hepes buffers pH7.0 may be usedin the formulations of the present invention. Any form of hepes suitablefor formulation and parenteral administration may be used in theformulation of the invention. The purity of hepes should be at least98%, at least 99%, or at least 99.5%. In a specific aspect, aformulation of the invention comprises 15 mM hepes buffer pH7.0.

It will be understood by one skilled in the art that the formulation ofthe invention may be isotonic or slightly hypotonic with human blood,i.e. the formulation of the invention has essentially the same or aslightly lower osmotic pressure as human blood. Such isotonic orslightly hypotonic formulation generally has an osmotic pressure fromabout 240 mOSm/kg to about 320 mOSm/kg, such as about 240 mOSm/kg orhigher, 250 mOSm/kg or higher or 260 mOSm/kg or higher.

Tonicity of a formulation is adjusted by the use of tonicity modifiers.“Tonicity modifiers” are those pharmaceutically acceptable inertsubstances that can be added to the formulation to provide anisotonicity of the formulation. A preferred tonicity modifier in theformulation of the invention are excipients. Preferred excipients foruse in the formulation of the invention may be selected from sugars,polyols and surfactants.

Accordingly, in another aspect, the formulation of the inventioncomprises an excipient. Preferred excipients include polyols and/orsugars. The polyol and/or sugar may be a monosaccharide such as glucoseor mannose, or a polysaccharide including disaccharides such as (withoutbeing limiting) sucrose and lactose, as well as sugar derivativesincluding sugar alcohols and sugar acids. Polyols and sugar alcoholsinclude (without being limiting) mannitol, xylitol, erythritol,threitol, sorbitol and glycerol. A non-limiting example of a sugar acidis L-gluconate. Other exemplary sugars include (without being limiting)trehalose, glycine, maltose, raffinose, etc. The concentration of theexcipient may range from about 1% to 10% (w:v), preferably from about2.5% to 10% (w:v), more preferably from about 5% to 10% (w:v), such ase.g. 5% (w:v), 7.5% (w:v), 8% or 10% (w:v). Throughout the presentinvention the concentration of the excipient will be given as % (w:v).In a preferred aspect, the formulation comprises sucrose, preferably ata concentration of about 5% to 10% (w:v), such as about 8% (w:v).

In another aspect, the formulation of the invention comprises asurfactant. A surfactant refers to a surface-active agent comprising ahydrophobic portion and a hydrophilic portion. In a preferred aspect,the surfactant is non-ionic. Certain exemplary non-ionic surfactantsinclude (without being limiting) PEG8000, and polysorbate, includingwithout being limiting, polysorbate 80 (TWEEN 80) and polysorbate 20(TWEEN 20), TRITON X-100, polyoxypropylene-polyoxyethylene esters(PLURONIC®), and NP-40. In a specific aspect, the surfactant is selectedfrom TWEEN (polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer. Theconcentration of the surfactant may range from about 0.001% to 1% (v:v)(preferably from about 0.001% to 0.1% (v:v), or 0.01% to 0.1% (v:v) suchas 0.001% (v:v), 0.005% (v:v), 0.01% (v:v), 0.02% (v:v), 0.05% (v:v),0.08% (v:v), 0.1% (v:v), 0.5% (v:v), or 1% (v:v) of the formulation,preferably 0.01% (v:v)). Throughout the present invention theconcentration of the surfactant will be given as % (v:v). In a specificembodiment, the surfactant is TWEEN (polysorbate) 20 or TWEEN(polysorbate) 80, which is at a concentration of 0.001% (v:v), 0.005%(v:v), 0.01% (v:v), 0.02% (v:v), 0.05% (v:v), 0.08% (v:v), 0.1% (v:v),0.5% (v:v) or 1% (v:v) of the formulation, preferably 0.01% (v:v).

In a preferred aspect, the formulation of the present inventioncomprises an aqueous carrier having a pH of 5.5 to 8.0 and a polypeptideas defined above (“polypeptide of the invention”) comprising one or moresingle variable domains at a concentration of 1 mg/mL to 200 mg/mL, saidformulation being formulated for administration to a human subject andsaid formulation further comprises at least two components selectedfrom:

-   -   a) A buffer at a concentration of 10 mM to 100 mM selected from        the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0,        MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;    -   b) An excipient at a concentration of 1% to 20%;    -   c) A surfactant at a concentration of 0.001% to 1% selected from        TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or poloxamers;        wherein said formulation has an inorganic salt concentration of        150 mM or lower.

In one aspect, in addition to the polypeptide of the invention, theformulation of the present invention may comprise at least an aqueouscarrier having a pH of 5.5 to 8.0, a buffer selected from the groupconsisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,succinate pH 6.0-6.5 and acetate pH 5.5-6.0, and an excipient.Preferably the buffer is histidine pH 6.0-6.5 or hepes pH 7.0. Mostpreferably the buffer is histidine pH 6.5 or histidine pH 6.0. Thebuffer preferably has a concentration ranging from 1 mM to 100 mM, 5 mMto 100 mM, 5 mM to 75 mM, 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25 mM,10 mM to 20 mM. In a specific aspect, the concentration of buffer in theformulations of the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM,25 mM, 50 mM, 75 mM, or 100 mM. In a preferred aspect, the concentrationis between 10 and 20 mM, such as 10 mM or 15 mM. In a specific aspect, aformulation of the invention comprises 15 mM histidine buffer pH 6.5. Inanother specific aspect, a formulation of the invention comprises 10 mMhistidine buffer pH 6.0. Preferred excipients are polyols such asmannitol, sorbitol, etc., saccharides such as e.g. sucrose, mannose,trehalose, etc. The concentration of the excipient may range from about1% to 20%, preferably from about 2.5% to 15%, more preferably from about5% to 10%, such as e.g. 5%, 7.5%, 8% or 10%. In a preferred aspect, theformulation comprises sucrose, preferably at a concentration of about 5%to 10%, such as about 8% or about 10%. Accordingly, a preferredformulation of the invention may comprise 15 mM histidine pH 6.5 and 8%sucrose. Another preferred formulation of the invention may comprise 10mM histidine pH 6.0 and 10% sucrose.

In another aspect, in addition to the polypeptide of the invention, theformulation of the present invention may comprise at least an aqueouscarrier having a pH of 5.5 to 8.0, a buffer selected from the groupconsisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,succinate pH 6.0-6.5 and acetate pH 5.5-6.0, and a surfactant.Preferably the buffer is selected from histidine pH 6.0-6.5 or hepes pH7.0. Most preferably the buffer is histidine pH 6.5 or histidine pH 6.0.The buffer preferably has a concentration ranging from 1 mM to 100 mM, 5mM to 100 mM, 5 mM to 75 mM, 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25mM, 10 mM to 20 mM. In a specific aspect, the concentration of buffer inthe formulation of the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20mM, 25 mM, 50 mM, 75 mM, or 100 mM. In a preferred aspect, theconcentration is between 10 and 20 mM, such as 10 mM or 15 mM. In aspecific aspect, a formulation of the invention comprises 15 mMhistidine buffer pH 6.5. In another specific aspect, a formulation ofthe invention comprises 10 mM histidine buffer pH 6.0. The surfactantmay be selected from TWEEN (polysorbate) 20, TWEEN (polysorbate) 80 orpoloxamers. The concentration of the surfactant may range from about0.001% to 1% (preferably from about 0.001% to 0.1%, or 0.01% to 0.1%such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% ofthe formulation, preferably 0.01%). In a specific embodiment, thesurfactant is TWEEN (polysorbate) 20 or TWEEN (polysorbate) 80, which isat a concentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,0.5% or 1% of the formulation, such as e.g. 0.01% TWEEN (polysorbate) 80or 0.005% TWEEN (polysorbate) 80. Accordingly, a preferred formulationof the invention may comprise 15 mM histidine pH 6.5 and 0.01% TWEEN(polysorbate) 80. Another preferred formulation of the invention maycomprise 10 mM histidine pH 6.0 and 0.005% TWEEN (polysorbate) 80.

In yet another aspect, in addition to the polypeptide of the invention,the formulation of the present invention may comprise at least anaqueous carrier having a pH of 5.5 to 8.0, an excipient and asurfactant. Preferred excipients are polyols such as mannitol, sorbitol,etc., saccharides such as e.g. sucrose, mannose, trehalose, etc. Theconcentration of the excipient may range from about 1% to 20%,preferably from about 2.5% to 15%, more preferably from about 5% to 10%,such as e.g. 5%, 7.5%, 8% or 10%. In a preferred aspect, the formulationcomprises sucrose, preferably at a concentration of about 5% to 10%,such as about 8% or 10%. The surfactant may be selected from TWEEN(polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer. Theconcentration of the surfactant may range from about 0.001% to 1%(preferably from about 0.001% to 0.1%, or 0.01% to t 0.1% such as0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of theformulation, preferably 0.01%). In a specific embodiment, the surfactantis TWEEN (polysorbate) 20 or TWEEN (polysorbate) 80, which is at aconcentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%or 1% of the formulation, such as e.g. 0.01% TWEEN (polysorbate) 80 or0.005% TWEEN (polysorbate) 80. Accordingly, a preferred formulation ofthe invention may comprise 8% sucrose and 0.01% TWEEN (polysorbate) 80.Another preferred formulation of the invention may comprise 10% sucroseand 0.005% TWEEN (polysorbate) 80.

Accordingly, a formulation of the invention may, in addition to thepolypeptide of the invention, comprise for example:

-   -   a) A buffer selected from a histidine buffer pH 6.5, histidine        buffer pH 6.0 and hepes buffer pH 7.0 at a concentration of 10        mM to 100 mM; and    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.5, histidine        buffer pH 6.0 and hepes buffer pH 7.0 at a concentration of 10        mM to 100 mM; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.5, and hepes        buffer pH 7.0 at a concentration of 15 mM; and    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 15 mM; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 10 mM to 100 mM; and    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 8%.    -   or    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 8%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 10 mM to 100 mM; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.01%.    -   or    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.01%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM; and    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM to 100 mM; and    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 10%.    -   or    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 10%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.    -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM to 100 mM; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.005%.    -   or    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.005%.    -   or    -   a) Histidine buffer pH 6.5 or pH 6.0 at a concentration of 10 mM        to 100 mM; and    -   b) Sucrose at a concentration of 1% to 20%.    -   or    -   a) Histidine buffer pH 6.5 or pH 6.0 at a concentration of 10 mM        to 100 mM; and    -   c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.    -   or    -   b) Sucrose at a concentration of 1% to 20%; and    -   c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.    -   or    -   a) 15 mM histidine buffer pH 6.5; and    -   b) 8% sucrose.    -   or    -   a) 15 mM histidine buffer pH 6.5; and    -   c) 0.01% TWEEN (polysorbate) 80.    -   or    -   b) 8% sucrose; and    -   c) 0.01% TWEEN (polysorbate) 80.    -   or    -   a) 10 mM histidine buffer pH 6.0; and    -   b) 10% sucrose.    -   or    -   a) 10 mM histidine buffer pH 6.0; and    -   c) 0.005% TWEEN (polysorbate) 80.    -   or    -   b) 10% sucrose; and    -   c) 0.005% TWEEN (polysorbate) 80.

In a preferred aspect the formulation of the invention may comprise apolypeptide selected from SEQ ID NO's: 1 to 6. Accordingly theformulation of the invention may comprise:

-   -   a) 15 mM histidine buffer pH 6.5;    -   b) 8% sucrose; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).    -   or    -   a) 15 mM histidine buffer pH 6.5;    -   c) 0.01% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).    -   or    -   b) 8% sucrose;    -   c) 0.01% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).    -   or    -   a) 10 mM histidine buffer pH 6.0;    -   b) 10% sucrose; and    -   c) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).    -   or    -   a) 10 mM histidine buffer pH 6.0;    -   c) 0.005% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).    -   or    -   b) 10% sucrose;    -   c) 0.005% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).

In another preferred aspect, the formulation of the present inventioncomprises an aqueous carrier having a pH of 5.5 to 8.0 and a polypeptideas defined above (“polypeptide of the invention”) comprising one or moresingle variable domains at a concentration of 1 mg/mL to 200 mg/mL, saidformulation being formulated for administration to a human subject andcomprises the components selected from:

-   -   a) A buffer at a concentration of 10 mM to 100 mM selected from        the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0,        MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;    -   b) An excipient at a concentration of 1% to 20%; and    -   c) A surfactant at a concentration of 0.001% to 1% selected from        TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer;        wherein said formulation has an inorganic salt concentration of        150 mM or lower.

Accordingly in this preferred aspect, in addition to the polypeptide ofthe invention, the formulations of the present invention may comprise atleast an aqueous carrier having a pH of 5.5 to 8.0, a buffer selectedfrom the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MESpH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0, an excipient and asurfactant. Preferably the buffer is selected from histidine pH 6.0-6.5or hepes pH 7.0. Most preferably the buffer is histidine pH 6.5 orhistidine pH 6.0. The buffer preferably has a concentration ranging from1 mM to 100 mM, 5 mM to 100 mM, 5 mM to 75 mM, 5 mM to 50 mM, 10 mM to50 mM, 10 mM to 25 mM, 10 mM to 20 mM. In a specific aspect, theconcentration of buffer in the formulations of the invention is 1 mM, 2mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, or 100 mM. In apreferred aspect, the concentration is between 10 and 20 mM, such as 10mM or 15 mM. In a specific aspect, a formulation of the inventioncomprises 15 mM histidine buffer pH 6.5. In another specific aspect, aformulation of the invention comprises 10 mM histidine buffer pH 6.0.Preferred excipients are polyols such as mannitol, sorbitol, etc.,saccharides such as e.g. sucrose, mannose, trehalose, etc. Theconcentration of the excipient may range from about 1% to 20%,preferably from about 2.5% to 15%, more preferably from about 5% to 10%,such as e.g. 5%, 7.5%, 8% or 10%. In a preferred aspect, the formulationcomprises sucrose, preferably at a concentration of about 5% to 10%,such as about 8% or 10%. The surfactant may be selected from TWEEN(polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer. Theconcentration of the surfactant may range from about 0.001% to 1%(preferably from about 0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%,0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of theformulation, preferably 0.01% or 0.005%). In a specific embodiment, thesurfactant is TWEEN (polysorbate) 20 or TWEEN (polysorbate) 80, which isat a concentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,0.5% or 1% of the formulation, such as e.g. 0.01% TWEEN (polysorbate) 80or 0.005% TWEEN (polysorbate) 80. Accordingly, a preferred formulationof the invention may comprise 15 mM histidine pH 6.5, 8% sucrose and0.01% TWEEN (polysorbate) 80. Another preferred formulation of theinvention may comprise 10 mM histidine pH 6.0, 10% sucrose and 0.005%TWEEN (polysorbate) 80.

Accordingly, a formulation of the invention may, in addition to thepolypeptide of the invention, comprise for example:

-   -   a) A buffer selected from a histidine buffer pH 6.5, a histidine        buffer pH 6.0 and hepes buffer pH 7.0 at a concentration of 10        mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 15 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 8%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.01%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 15 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 8%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 8%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.01%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.5 and hepes        buffer pH 7.0 at a concentration of 15 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.01%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 10%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.005%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 10%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.001% to        1%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 10 mM to 100 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 10%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.005%.        -   or    -   a) A buffer selected from a histidine buffer pH 6.0 at a        concentration of 15 mM;    -   b) An excipient selected from sucrose, sorbitol, trehalose and        mannitol at a concentration of 1% to 20%; and    -   c) A surfactant selected from TWEEN (polysorbate) 80, TWEEN        (polysorbate) 20 or a poloxamer at a concentration of 0.005%.        -   or    -   a) Histidine buffer pH 6.5 at a concentration of 10 mM to 100        mM;    -   b) Sucrose at a concentration of 1% to 20%; and    -   c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.        -   or    -   a) 15 mM histidine buffer pH 6.5;    -   b) 8% sucrose; and    -   c) 0.01% TWEEN (polysorbate) 80.        -   or    -   a) Histidine buffer pH 6.0 at a concentration of 10 mM to 100        mM;    -   b) Sucrose at a concentration of 1% to 20%; and    -   c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.        -   or    -   a) 10 mM histidine buffer pH 6.0;    -   b) 10% sucrose; and    -   c) 0.005% TWEEN (polysorbate) 80.

In a preferred aspect the formulation of the invention may comprise apolypeptide selected from SEQ ID NO's: 1 to 6. Accordingly theformulation of the invention may comprise:

-   -   a) 15 mM histidine buffer pH 6.5;    -   b) 8% sucrose;    -   c) 0.01% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).        -   or    -   a) 10 mM histidine buffer pH 6.0;    -   b) 10% sucrose;    -   c) 0.005% TWEEN (polysorbate) 80; and    -   d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at a        concentration of 10 mg/ml).

The components present in the formulations of the invention have beenselected such that the polypeptides of the invention have a goodsolubility (as defined herein). Preferably, the polypeptides present inthe formulations of the present invention have a solubility of at least0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1.0 mM, at least 1.1mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, at least 1.5 mM,at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, atleast 2.0 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, atleast 2.4 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, atleast 2.8 mM, at least 2.9 mM, at least 3.0 mM, at least 3.2 mM, atleast 3.4 mM, at least 3.6 mM or more and/or at least 20 mg/ml, at least30 mg/mL, at least 40 mg/mL, at least 50 mg/mL, at least 60 mg/mL, atleast 65 mg/mL, at least 70 mg/mL, at least 80 mg/mL, at least 90 mg/mL,at least 100 mg/mL, at least 110 mg/mL, at least 120 mg/mL, at least 130mg/mL, at least 140 mg/mL, at least 150 mg/mL or even 200 mg/mL or moreas determined by the PEG exclusion method or by centrifugalultrafiltration. A very good solubility of the polypeptides of theinvention has been obtained with a formulation comprising a histidinebuffer pH 6.5 or with a formulation comprising TWEEN (polysorbate) 80.Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastone of:

-   -   a) A histidine buffer pH 6.5 at a concentration of 10 mM to 100        mM (preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such        as 15 mM);    -   c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%        (preferably from about 0.001% to 0.1%, or 0.01% to 0.1% such as        0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of        the formulation, preferably 0.01%);        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein the solubility of the polypeptide is at least at least        20 mg/mL, at least 50 mg/mL, preferably at least 90 mg/mL, at        least 120 mg/mL, at least 150 mg/mL or even 200 mg/mL or more as        determined by the PEG exclusion method or by centrifugal        ultrafiltration. In a preferred aspect, the formulation        comprises a histidine buffer pH 6.5 at a concentration of 10 mM        to 100 mM (preferably 10 mM to 50 mM, more preferably 10 to 20        mM, such as 15 mM) and TWEEN (polysorbate) 80 at a concentration        of 0.001% to 1% (preferably from about 0.001% to 0.1%, or 0.01%        to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%,        0.1%, 0.5%, or 1% of the formulation, preferably 0.01%).

Apart from this and/or in addition, the polypeptides of the inventionpresent in the formulation of the invention should preferably have amelting temperature of at least 59° C. or more (such as 59.5° C. ormore), preferably at least 60° C. or more (such as 60.5° C. or more),more preferably at least 61° C. or more (such as 61.5° C. or more) or atleast 62° C. or more (such as 62.5° C. or more), most preferably atleast 63° C. or more (such as 63.5° C. or more) as measured by thethermal shift assay (TSA) and/or differential scanning calorimetry(DSC).

Without being limiting, melting point determination can be done by thefluorescence-based thermal shift assay which is based on the fact thatupon thermal unfolding the hydrophobic regions of proteins, usuallyhidden in the core of the protein fold, become accessible for binding toa hydrophobic fluorescent dye. The fluorescence emission of this dye isquenched in aqueous solution, whereas upon binding to the hydrophobicpatches of an unfolded protein a sharp increase in the fluorescenceyield of the probe is observed. Temperature induced unfolding istypically a two-state process with a sharp transition between the foldedand unfolded state, where the melting temperature (Tm) is defined as thetemperature at which half of the protein is in the unfolded state, i.e.the first derivative of the fluorescence signal upon gradual heating ofthe sample is plotted and the observed peak (or peaks when multipledomains and/or variants of the same domain are present) represents themelting temperature. The thermal shift assay can be performed in atypical real-time PCR instrument where melting curves can be recordedaccurately in high-throughput mode with only small quantities of proteinrequired.

During a differential scanning calorimetry experiment the sample isheated at a constant rate in an adiabatic environment (ΔT=0). The energyrequired to keep the temperature difference between a reference and thesample cell at zero is measured and yields the heat capacity as afunction of temperature (Cp(T)). The temperature corresponding to themaximum heat capacity represents the melting temperature (T_(m)). If thetemperature dependent unfolding process is reversible otherthermodynamic parameters such as the unfolding enthalpy (ΔH_(unfolding))can be determined.

Increased melting temperatures have been observed for the polypeptidesof the invention when present in a formulation with a pH of about 6.0 to8.0, preferably 6.2 to 7.5, more preferably 6.5 to 7.5, most preferably6.5-7.0. Increased melting temperature have also been obtained for thepolypeptides of the invention when present in a formulation thatcomprise hepes pH 7.0, histidine pH 6.0-6.5, MES pH 6.0 or acetate pH6.0, or a formulation that comprises an excipient, preferably asaccharides and/or polyol such as mannitol, trehalose, sorbitol orsucrose. Accordingly, the present invention relates to a formulationcomprising an aqueous carrier at a pH of 6.0 to 8.0 and a polypeptidecomprising one or more single variable domains, said formulation beingformulated for administration to a human subject, wherein saidformulation further comprises at least one of:

-   -   a) A buffer at a concentration of 10 mM to 100 mM (preferably 10        mM to 50 mM, more preferably 10 to 20 mM, such as 15 mM or 10        mM) selected from the group consisting of histidine pH 6.0-6.5,        hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate        pH 5.5-6.0;    -   b) An excipient, preferably a saccharide and/or polyol such as        mannitol, sorbitol, trehalose or sucrose at a concentration of        1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%,        such as 5%, 7.5%, 8% or 10%).        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein the melting temperature of the polypeptide of the        invention is at least 59° C. or more (such as 59.5° C. or more),        preferably at least 60° C. or more (such as 60.5° C. or more),        more preferably at least 61° C. or more (such as 61.5° C. or        more) or at least 62° C. or more (such as 62.5° C. or more),        most preferably at least 63° C. or more (such as 63.5° C. or        more) as measured by the thermal shift assay (TSA) and/or        differential scanning calorimetry (DSC).

In a preferred aspect, the formulation comprises a buffer at aconcentration of 10 mM to 100 mM (preferably 10 mM to 50 mM, morepreferably 10 to 20 mM, such as 15 mM) selected from the groupconsisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,succinate pH 6.0-6.5 and acetate pH 5.5-6.0, and an excipient,preferably a saccharide and/or polyol such as mannitol, sorbitol,trehalose or sucrose at a concentration of 1% to 20% (preferably 2.5% to15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%).

Apart from this and/or in addition, the formulation of the presentinvention exhibits stability under at least one or more of the followingstress conditions:

-   -   multiple (up to 10) freeze/thaw cycles;    -   storage at a temperature of 2-8° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   storage at a temperature of 25±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   storage at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   mechanical stress.

Preferably the formulation of the invention is stable under one or moreof the following forms of mechanical stress:

-   -   shaking the formulation during 10 s to 1 min;    -   pushing the formulation through a needle (25G, preferably 26G,        more preferably 27G, even more preferably 28G, most preferably        29G or more) with a syringe (the syringe used can be any        commercially available syringe, such as e.g. a 1 mL, 2 mL, 3 mL,        4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL up to 50 mL syringe);    -   rotating for two days at 10 rpm; and/or    -   stirring for 1 hour at room temperature and/or 4-48 hours at        4° C. at at least 10 rpm (such as 50 rpm, 100 rpm or more).

Preferably, the formulation of the present invention is stable undermore than one of the above stress conditions, such as at least two, atleast three, at least four, at least five, at least six, at least sevenor most preferably under all of the above stress conditions.

In one aspect, the formulation of the invention exhibits stability underone or more forms of mechanical and/or shear stress. Mechanical stressas used in the present invention can be any form of external forceapplied on the formulation that may affect the stability of thepolypeptide present in the formulation. Without being limiting, themechanical stress applied to the solution include shear stress, stirstress, shake stress, rotation stress, etc. The formulation of theinvention may for example be shaken during at least 10 s, 20 s, 30 s, 40s, 50 s up to 1 minute or more. The formulation of the invention may bepushed through a syringe (the syringe used can be any commerciallyavailable syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL,20 mL, 30 mL, 40 mL up to 50 mL syringe) with needle once, twice, threetimes, four times, five times up to 10 times or more. Preferably theneedle has a size of 25G (such as 26G, 27G, 28G, 29G, 30G) or more. Morepreferably the size of the needles is 27G or more. In addition oralternatively, the formulation of the invention may be rotated for 1hour, 2 hours, 6 hours, 12 hours, 1 day up to two days or more at 10rpm. The formulation of the invention may be stirred for 1 hour at roomtemperature and/or 4 hours, 8 hours, 12 hours, 24 hours or even 48 hoursor more at 2-8° C. The speed of rotation is preferably above 10 rpm,such as e.g. 50 rpm, 100 rpm or more.

The stability of the formulation under mechanical stress can be assessede.g. by visual inspection of the formulation or by measurement of theOD320/OD280 ratio. Preferably the OD320/OD280 ratio is 0.05 or lower,such as 0.01 or 0.005.

A good stability of the polypeptides of the invention under mechanicalstress has been obtained with a formulation comprising an excipient,preferably a saccharide and/or polyol such as mannitol or sucrose orcomprising TWEEN (polysorbate) 80. Accordingly, the present inventionrelates to a formulation comprising an aqueous carrier and a polypeptidecomprising one or more single variable domains, said formulation beingformulated for administration to a human subject, wherein saidformulation further comprises at least one of:

-   -   b) An excipient, preferably a saccharide and/or polyol such as        mannitol or sucrose at a concentration of 1% to 20% (preferably        2.5% to 10%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or        10%);    -   c) A surfactant at a concentration of 0.001% to 1% (preferably        from about 0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%,        0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the        formulation, preferably 0.01%) selected from TWEEN (polysorbate)        20, TWEEN (polysorbate) 80 or a poloxamer,        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein the polypeptide is stable under mechanical stress as        determined by OD320/OD280 ratio measurement. In a preferred        aspect, the formulation comprises an excipient, preferably a        saccharide and/or polyol such as mannitol or sucrose at a        concentration of 1% to 20% (preferably 2.5% to 15%, more        preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and a        surfactant at a concentration of 0.001% to 1% (preferably about        0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%,        0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation,        preferably 0.01% or 0.005%) selected from TWEEN (polysorbate) 20        and TWEEN (polysorbate) 80. A preferred formulation comprises 8%        sucrose and 0.01% TWEEN (polysorbate) 80. Another preferred        formulation comprises 10% sucrose and 0.005% Tween 80.

In a specific aspect, the formulation of the invention comprises anexcipient, preferably a saccharide and/or polyol such as mannitol orsucrose at a concentration of 1% to 20% (preferably 2.5% to 15%, morepreferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or TWEEN(polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer (at aconcentration ranging from about 0.001% to 0.1%, or 0.01% to 0.1% suchas 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of theformulation, preferably 0.01%) and is characterized that thepolypeptides present in the formulation are stable under mechanicalstress.

In a preferred aspect, the formulation of the invention comprises anexcipient, preferably a saccharide and/or polyol such as mannitol orsucrose at a concentration of 1% to 20% (preferably 2.5% to 15%, morepreferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or TWEEN(polysorbate) 80 (at a concentration of 0.001%, 0.005%, 0.01%, 0.02%,0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or0.005%) and is characterized that the polypeptides present in theformulation of the invention are stable when shaking the formulationduring at least 10 s, 20 s, 30 s, 40 s, 50 s up to 1 minute of more. Inanother preferred aspect, the formulation of the invention comprises anexcipient, preferably a saccharide and/or polyol such as mannitol orsucrose at a concentration of 1% to 20% (preferably 2.5% to 15%, morepreferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or TWEEN(polysorbate) 80 (at a concentration of 0.001%, 0.005%, 0.01%, 0.02%,0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or0.005%) and is characterized that the polypeptides present in theformulation of the invention are stable when pushing the formulationthrough a syringe (the syringe used can be any commercially availablesyringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL, 30mL, 40 mL up to 50 mL syringe) with needle size of 25G or more (such as26G, 27G, 28G, 29G, 30G or more, preferably 27G or more) once, twice,three times, four times, five times up to 10 times or more. In anotherpreferred aspect, the formulation of the invention comprises anexcipient, preferably a saccharide and/or polyol such as mannitol orsucrose at a concentration of 1% to 20% (preferably 2.5% to 15%, morepreferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or TWEEN(polysorbate) 80 (at a concentration of 0.001%, 0.005%, 0.01%, 0.02%,0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or0.005%) and is characterized that the polypeptides present in theformulation of the invention are stable when rotating the formulationfor 1 hour, 2 hours, 6 hours, 12 hours, 1 day up to two days or more at10 rpm. In another preferred aspect, the formulation of the inventioncomprises an excipient, preferably a saccharide and/or polyol such asmannitol or sucrose at a concentration of 1% to 20% (preferably 2.5% to15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/orTWEEN (polysorbate) 80 (at a concentration of 0.001%, 0.005%, 0.01%,0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably0.01% or 0.005%) and is characterized that the polypeptides present inthe formulation of the invention are stable when stirring theformulation for 1 hour at room temperature and/or 4 hours, 8 hours, 12hours, 24 hours or even 48 hours or more at 2-8° C. at at least 10 rpm(such as 50 rpm, 100 rpm or more).

A “freeze/thaw cycle” or “F/T cycle” is defined as the freezing of asample in a freezer (−20±5° C.) or ultrafreezer (below −64° C. (such ase.g. at −80° C.)) until solid, followed by thawing at room temperatureuntil all ice crystals have visually disappeared. In one aspect, theformulation of the invention exhibits stability under multiple (up to10) freeze/thaw cycles. The formulation of the invention may exhibitstability under at least 1, at least 2, at least 3, at least 5 to up toat least 10 freeze/thaw cycles, such as e.g. 10 cycles at −20° C., 2cycles at −80° C.+1 cycle at −20° C. or 2 cycles at −80° C.+6 cycles at−20° C.

In yet another aspect, the formulation of the invention exhibitsstability when stored at a temperature of 5±5° C. The formulation of theinvention may exhibit stability when stored at a temperature of 5±5° C.for at least 2 weeks, 3 weeks, 4 weeks, 8 weeks, 10 weeks, up to 3months, 6 months, 11 months, 1 year, 1.5 year or even 2 years and more.

In yet another aspect, the formulation of the invention exhibitsstability when stored at a temperature of 25±5° C. The formulation ofthe invention may exhibit stability when stored at a temperature of25±5° C. for at least 2 weeks, 3 weeks, 4 weeks, at least 5 weeks, atleast 8 weeks, at least 10 weeks, at least 3 months, at least 6 months,at least 1 year, 1.5 year or even 2 years or more.

In yet another aspect, the formulation of the invention exhibitsstability when stored at a temperature of 37±5° C. The formulation ofthe invention may exhibit stability when stored at a temperature of37±5° C. for at least 2 weeks, 3 weeks, 4 weeks, at least 5 weeks, atleast 8 weeks, at least 10 weeks, at least 3 months, at least 6 months,at least 1 year, 1.5 year or even 2 years or more.

As is known to one skilled in the art, the temperatures indicated inthis text can be subject to normal variations.

Preferably, in those formulations that are stable under one or more ofthe above stress conditions:

-   -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms pyroglutamate at the        N-terminal glutamic acid (e.g. as assessed by RP-HPLC) during        storage under (one of the above) stress conditions, such as e.g.        at a temperature of 37±5° C. up to at least 2 weeks (preferably        at least 3 weeks, at least 5 weeks, at least 8 weeks, at least        10 weeks, at least 3 months, at least 6 months, at least 1 year,        1.5 year or even 2 years or more);    -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms dimers (e.g. as assessed by        SE-HPLC) during storage under (one of the above) stress        conditions, such as e.g. at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more);    -   at least 80% (at least 85%, at least 90%, at least 95%, at least        98%, at least 99%, or at least 99.5%) of the polypeptides of the        invention retain their binding activity (e.g. as assessed by        ELISA and/or BIACORE) to at least one of their (preferably to        all of their) targets after storage under (one of the above)        stress conditions, such as e.g. at a temperature of 37±5° C. up        to at least 2 weeks (preferably at least 3 weeks, at least 5        weeks, at least 8 weeks, at least 10 weeks, at least 3 months,        at least 6 months, at least 1 year, 1.5 year or even 2 years or        more) compared to the binding activity prior to the stress        condition.

Stability of the formulations can be assessed by various analyticaland/or immunological methods known in the art. The protein content ofthe polypeptides of the invention can, for example be detected byspectrophotometrical methods.

SDS-PAGE allows the visualization of the polypeptides in a given samplevia direct staining. SDS-PAGE is used to separate proteins according totheir size. Both reducing and non-reducing SDS-PAGE analysis can beperformed.

The molecular size distribution and the relative amounts of polypeptideof the invention and protein impurities can be determined by SizeExclusion High Performance Liquid Chromatography (SE-HPLC). The relativeamount of a specific protein impurity, expressed as area %, can becalculated by dividing the peak area corresponding to the impurity bythe total integrated area. SE-HPLC methods are known to the skilledperson and are also described in the Example section.

Reversed Phase High Performance Liquid chromatography (RP-HPLC)separates molecules with respect to differences in hydrophobicity and isbased on the reversible interaction between the molecule and thehydrophobic stationary phase. In this assay a ZORBAX 300SB-C3 column(Agilent Technologies, Palo Alto, US) can be used. The relative amountof a specific protein impurity, expressed as area %, can be calculatedby dividing the peak area corresponding to the impurity by the totalintegrated area. RP-HPLC methods are known to the skilled person and arealso described in the Example section.

Polypeptides of the invention and their charge variants can be separatedby Ion Exchange High Performance Liquid Chromatography (IEX-HPLC). Alsopotential impurities can be detected with this method. The relativeamount of a specific protein impurity, expressed as area %, can becalculated by dividing the peak area corresponding to the impurity bythe total integrated area. IEX-HPLC methods are known to the skilledperson and are also described in the Example section.

The polypeptides present in the formulation of the invention preferablydo not form pyroglutamate at the N-terminal glutamic acid. The formationof pyroglutamate in the sample can e.g. be measured by RP-HPLC. Forexample, analysis by RP-HPLC of a formulation containing SEQ ID NO: 4after storage for 10 weeks at a temperature of 37° C., showed theformation of pyroglutamate as a separate peak at 18-19 minutes.Preferably in the formulation of the invention, less than 10% (morepreferably less than 5%, even more preferably less than 3%, mostpreferably less than 1%) of the polypeptides form pyroglutamate at theN-terminal glutamic acid (e.g. as assessed by RP-HPLC) under one or moreof the above stress conditions.

Little to no pyroglutamate formation of the polypeptides of theinvention has been observed in formulations with a pH of 7.0 or lower,preferably 6.5 or lower, such as 6.5, 6.0 or 5.5, in e.g. histidinebuffers and acetate buffers. Accordingly, the present invention relatesto a formulation comprising an aqueous carrier at a pH of 7.0 or lowerand a polypeptide comprising one or more single variable domains, saidformulation being formulated for administration to a human subject,wherein said formulation further comprises:

-   -   a) A buffer at a concentration of 10 mM to 100 mM (preferably 10        mM to 50 mM, more preferably 10 to 20 mM, such as 15 mM or 10        mM) selected from the group consisting of histidine pH 6.0-6.5        and acetate pH 5.5-6.0,        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein less than 10% (preferably less than 8%, more preferably        less than 7%, most preferably less than 5%) of the polypeptides        forms pyroglutamate at the N-terminal glutamic acid during one        or more of the above stress conditions (such as during storage        at a temperature of 37±5° C. up to at least 2 weeks (preferably        at least 3 weeks, at least 5 weeks, at least 8 weeks, at least        10 weeks, at least 3 months, at least 6 months, at least 1 year,        1.5 year or even 2 years or more)), the % of pyroglutamate as        measured by RP-HPLC. In a preferred aspect, the formulation        comprises a histidine buffer pH 6.5 at 15 mM. In another        preferred aspect, the formulation comprises a histidine buffer        pH 6.0 at 10 mM.

The polypeptides present in the formulation of the invention alsopreferably do not form dimers. The formation of dimers in the sample cane.g. be measured by SE-HPLC. For example, analysis in SE-HPLC of aformulation containing SEQ ID NO: 4 after storage for 10 weeks at atemperature of 37° C., showed the formation of a separate peak elutingat an apparent molecular weight of 44 kDa in comparison with molecularweight markers, while the monomeric polypeptide eluted between the 44and 17 kDa molecular weight markers. This separate peak at 44 kDarepresented a dimeric form of SEQ ID NO: 4. Preferably in theformulation of the invention, less than 10% (more preferably less than5%, even more preferably less than 3%, most preferably less than 1%) ofthe polypeptides forms dimers (e.g. as assessed by SE-HPLC) duringstorage under one or more of the above stress conditions.

Little to no dimer formation of the polypeptides of the invention hasbeen observed in formulations with a histidine buffer or an acetatebuffer and in formulations that comprise an excipient, preferably asaccharide and/or polyol such as mannitol, trehalose, sorbitol orsucrose. Accordingly, the present invention relates to a formulationcomprising an aqueous carrier and a polypeptide comprising one or moresingle variable domains, said formulation being formulated foradministration to a human subject, wherein said formulation furthercomprises at least one of:

-   -   a) A buffer at a concentration of 10 mM to 100 mM (preferably 10        mM to 50 mM, more preferably 10 to 20 mM, such as 15 mM or 10        mM) selected from the group consisting of histidine pH 6.0-6.5        and acetate pH 5.5-6.0;    -   b) An excipient, preferably a saccharide, a non-reducing sugar        and/or polyol such as mannitol, trehalose, sorbitol or sucrose        at a concentration of 1% to 20% (preferably 2.5% to 15%, more        preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%),        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein less than 10% (preferably less than 8%, more preferably        less than 7%, most preferably less than 5%) of the polypeptides        forms dimers during one or more of the above stress conditions        (such as during storage at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more)), the        % of dimers as measured by SE-HPLC. In a preferred aspect, the        formulation comprises a histidine buffer pH 6.5 at 15 mM. In        another preferred aspect, the formulation comprises a histidine        buffer pH 6.0 at 10 mM. In a preferred aspect, the formulation        comprises a buffer at a concentration of 10 mM to 100 mM        (preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as        15 mM or 10 mM) selected from the group consisting of histidine        pH 6.0-6.5 and acetate pH 5.5-6.0 and an excipient, preferably a        saccharide, non-reducing sugar and/or polyol such as mannitol,        trehalose, sorbitol or sucrose at a concentration of 1% to 20%        (preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%,        7.5%, 8% or 10%), such as e.g. 15 mM histidine pH 6.5 and 8%        sucrose; or 10 mM histidine pH 6.0 and 10% sucrose.

Preferably in the formulation of the invention, less than 10% (morepreferably less than 5%, even more preferably less than 3%, mostpreferably less than 1%) of the polypeptides forms pyroglutamate at theN-terminal glutamic acid (e.g. as assessed by RP-HPLC) and less than 10%(more preferably less than 5%, even more preferably less than 3%, mostpreferably less than 1%) of the polypeptides forms dimers (e.g. asassessed by SE-HPLC) during storage under one or more of the abovestress conditions.

Apart from this and/or in addition, the formulation of the presentinvention should show only low to undetectable levels of aggregationeven during storage under one or more of the above stress conditions.For example, in the formulation of the invention, no more than 5%, nomore than 4%, no more than 3%, no more than 2%, no more than 1%, andmost preferably no more than 0.5% of the polypeptides forms an aggregateafter storage under one or more of the above stress conditions.

Aggregation as used in the present invention means the development ofhigh molecular weight aggregates, i.e. aggregates with an apparentmolecular weight in SE-HPLC analysis of more/higher than the molecularweight observed for dimers. As described above, 44 kDa is the apparentmolecular weight observed in SE-HPLC analysis for dimers of SEQ ID NO:4, 36-38 kDa is the apparent molecular weight observed in SE-HPLCanalysis for dimers of SEQ ID NO's: 1-3 and 36 kDa is the apparentmolecular weight observed in SE-HPLC analysis for dimers of SEQ ID NO:5. Aggregation can be assessed by various methods known in the art.Without being limiting, examples include SE-HPLC, analyticalultracentrifugation, dynamic light scattering, subvisible particlecounting and OD320/OD280 measurement.

In an analytical ultracentrifuge, a sample being spun can be monitoredin real time through an optical detection system, using ultravioletlight absorption and/or interference optical refractive index sensitivesystem. This allows the operator to observe the evolution of the sampleconcentration versus the axis of rotation profile as a result of theapplied centrifugal field. With modern instrumentation, theseobservations are electronically digitized and stored for furthermathematical analysis. Two kinds of experiments are commonly performedon these instruments: sedimentation velocity experiments andsedimentation equilibrium experiments.

Sedimentation velocity experiments aim to interpret the entiretime-course of sedimentation, and report on the shape and molar mass ofthe dissolved macromolecules, as well as their size-distribution(Perez-Ramirez and Steckert (2005) Therapeutic Proteins: Methods andProtocols. C. M. Smales and D. C. James, Eds. Vol. 308: 301-318. HumanaPress Inc, Totowa, N.J., US.). The size resolution of this method scalesapproximately with the square of the particle radii, and by adjustingthe rotor speed of the experiment size-ranges from 100 Da to 10 GDa canbe covered. Sedimentation velocity experiments can also be used to studyreversible chemical equilibria between macromolecular species, by eithermonitoring the number and molar mass of macromolecular complexes, bygaining information about the complex composition from multi-signalanalysis exploiting differences in each components spectroscopic signal,or by following the composition dependence of the sedimentation rates ofthe macromolecular system, as described in Gilbert-Jenkins theory.

Sedimentation equilibrium experiments are concerned only with the finalsteady-state of the experiment, where sedimentation is balanced bydiffusion opposing the concentration gradients, resulting in atime-independent concentration profile. Sedimentation equilibriumdistributions in the centrifugal field are characterized by Boltzmanndistributions. This experiment is insensitive to the shape of themacromolecule, and directly reports on the molar mass of themacromolecules and, for chemically reacting mixtures, on chemicalequilibrium constants.

The kinds of information that can be obtained from an analyticalultracentrifuge include the gross shape of macromolecules, theconformational changes in macromolecules, and size distributions ofmacromolecular samples. For macromolecules, such as proteins, that existin chemical equilibrium with different non-covalent complexes, thenumber and subunit stoichiometry of the complexes and equilibriumconstant constants can be studied. (see also Scott D. J., Harding S. E.and Rowe A. J. Analytical Ultracentrifugation Techniques and Methods,RSC Publishing)

Dynamic light scattering (also known as Photon Correlation Spectroscopyor quasi-elastic light scattering) is a technique in physics, which canbe used to determine the size distribution profile of small particles insolution. When a beam of light passes through a colloidal dispersion,the particles or droplets scatter some of the light in all directions.When the particles are very small compared with the wavelength of thelight, the intensity of the scattered light is uniform in all directions(Rayleigh scattering); for larger particles (above approximately 250 nmdiameter), the intensity is angle dependent (Mie scattering). If thelight is coherent and monochromatic, as from a laser for example, it ispossible to observe time-dependent fluctuations in the scatteredintensity using a suitable detector such as a photomultiplier capable ofoperating in photon counting mode.

These fluctuations arise from the fact that the particles are smallenough to undergo random thermal (Brownian) motion and the distancebetween them is therefore constantly varying. Constructive anddestructive interference of light scattered by neighbouring particleswithin the illuminated zone gives rise to the intensity fluctuation atthe detector plane which, as it arises from particle motion, containsinformation about this motion. Analysis of the time dependence of theintensity fluctuation can therefore yield the diffusion coefficient ofthe particles from which, via the Stokes Einstein equation, knowing theviscosity of the medium, the hydrodynamic radius or diameter of theparticles can be calculated. (see also Berne B. J. and Pecora R. DynamicLight Scattering With Applications to Chemistry, Biology and Physics,Dover Publications)

Aggregation can also be measured by the PAMAS SVSS-C(Small VolumeSyringe System-C) instrument (PArtikelMess—und AnalyseSysteme GMBH),which is a particle size distribution analyzer for low viscous fluids.It uses the principle of light obscuration to detect sub-visibleparticles in the size range 1 μm-200 μm. The validationcriteria/specified limits of the European Pharmacopoeia (EP<2.9.19Particulate Contamination: sub-visible particles) for small and largevolume parenterals are defined by the total counts per container:

For particles>10 μm, no more than 6000 counts per container

For particles>25 μm, no more than 600 counts per container

The OD320/OD280 ratio is also a measure for turbidity or the presence ofparticulates in the sample. In a preferred aspect, the OD320/OD280 ratioof the formulation of the invention should be 0.05 or lower, preferably0.01 or lower, such as 0.005 or lower.

The tendency for aggregate formation of a polypeptide in a certainformulation can also be measured by elastic light scattering. Elasticlight scattering can be measured in a spectrofluorometer (e.g.excitation and emission wavelength 500 nm) by temperature-induceddenaturation as measured e.g. at an angle of 90°. Preferably the maximumscatter will stay within the absorption detection limit. The scattershould be 1000 abs. or lower, preferably 750 abs or lower, such as 500abs or lower.

No particulate formation has been observed in formulations comprising ahistidine buffer pH 6.0-6.5 under different stress conditions (such ase.g. storage at a temperature of 5±5° C. up to up to at least 2 weeks(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks, atleast 10 weeks, at least 3 months, at least 6 months, at least 1 year,1.5 year or even 2 years or more) or storage at a temperature of 37±5°C. up to at least 2 weeks (preferably at least 3 weeks, at least 5weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at least6 months, at least 1 year, 1.5 year or even 2 years or more)).Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises:

-   -   a) A histidine buffer pH 6.0-6.5 at a concentration of 10 mM to        100 mM (preferably 10 mM to 50 mM, more preferably 10 to 20 mM,        such as 15 mM),        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein no particulates are present under one or more of the        above stress conditions (e.g. when stored at a temperature of        5±5° C. up to up to at least 2 weeks (preferably at least 3        weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at        least 3 months, at least 6 months, at least 1 year, 1.5 year or        even 2 years or more) or when stored at a temperature of        37±5° C. up to at least 2 weeks (preferably at least 3 weeks, at        least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3        months, at least 6 months, at least 1 year, 1.5 year or even 2        years or more)) as measured by OD320/OD280 ratio measurement        and/or elastic light scattering. In a preferred aspect the        formulation comprises a histidine buffer pH 6.5 at 15 mM. In        another preferred aspect the formulation comprises a histidine        buffer pH 6.0 at 10 mM.

Apart from this and/or in addition, the formulation of the presentinvention shows only low to undetectable levels of fragmentation and/ordegradation even during storage under one ore more of the above stressconditions. Fragmentation and degradation can be measured e.g. bySE-HPLC and/or RP-HPLC. For example, analysis in SE-HPLC of aformulation containing SEQ ID NO: 4 after storage for 10 weeks at atemperature of 37° C., showed the formation of some minor postpeaks,representing degradation products of SEQ ID NO: 4. For example, analysisby RP-HPLC of a formulation containing SEQ ID NO: 4 after storage for 10weeks at a temperature of 37° C., showed the formation of some minorpeaks at 8-9 minutes, representing degradation products. Preferably inthe formulation of the invention, no more than 5%, no more than 4%, nomore than 3%, no more than 2%, no more than 1%, no more than 0.5%, nomore than 0.1%, no more than 0.05%, and most preferably no more than0.01% of the polypeptides shows degradation and/or fragmentation afterstorage under one or more of the above stress conditions.

The techniques of static light scattering (SLS), tangential flowfiltration (TFF), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea-induced protein unfolding techniques,intrinsic tryptophan fluorescence and/or 1-anilino-8-naphthalenesulfonicacid (ANS) protein binding can also be used to assess the physicalproperties and stability of polypeptides.

Apart from this and/or in addition, the formulation of the presentinvention shows very little to no loss of potency and/or biologicalactivity of their polypeptides, even during storage under one ore moreof the above stress conditions.

The potency and/or biological activity of a biological describes thespecific ability or capacity of said biological to achieve a definedbiological effect. The potency and biological activities of thepolypeptides of the invention can be assessed by various assaysincluding any suitable in vitro assay, cell-based assay, in vivo assayand/or animal model known per se, or any combination thereof, dependingon the specific disease or disorder involved. Suitable in vitro assayswill be clear to the skilled person, and for example include ELISA; FACSbinding assay; BIACORE; competition binding assay (AlphaScreen®, PerkinElmer, Massachusetts, USA; FMAT); TRAP assay (osteoclast differentiationassay; Rissanen et al. 2005, J. Bone Miner. Res. 20, Suppl. 1: S256);NF-kappaB reporter gene assay (Mizukami et al. 2002, Mol. Cell. Biol.22: 992-1000). For example, SEQ ID NO: 4 interacts with RANKL and blocksthe interaction of this ligand with RANK, thereby preventingsignalization through this receptor. SEQ ID NO's: 1 to 3 interact withIL-6R and block the interaction of this receptor with IL-6. SEQ ID NO's:5 and 6 interact with IL-23 and block the interaction of this ligandwith its receptor. The potency of SEQ ID NO's: 1 to 6 for blocking therespective ligand/receptor interaction can be determined, e.g. by ELISA,BIACORE, AlphaScreen®.

For example, in one embodiment, BIACORE kinetic analysis uses SurfacePlasmon Resonance (SPR) technology to monitor macromolecularinteractions in real time and is used to determine the binding on andoff rates of polypeptides of the formulation of the invention to theirtarget. BIACORE kinetic analysis comprises analyzing the binding anddissociation of the target from chips with immobilized polypeptides ofthe invention on their surface. A typical BIACORE kinetic study involvesthe injection of 250 μL of polypeptide reagent at varying concentrationin HBS buffer containing 0.005% TWEEN (polysorbate) 20 over a sensorchip surface, onto which has been immobilized the antigen. In theBIACORE 3000 system, the ligand is immobilized on carboxymethylateddextran over a gold surface, while the second partner (analyte) iscaptured as it flows over the immobilized ligand surface. Theimmobilized ligands are remarkably resilient and maintain theirbiological activity. The bound analytes can be stripped from theimmobilized ligand without affecting its activity to allow many cyclesof binding and regeneration on the same immobilized surface. Interactionis detected in real time via SPR and at high sensitivity. Because thesame affinity may reflect different on-rates and off-rates, thisinstrument excels over most other affinity measuring methods in that itmeasures on-rates (ka) and off-rates (kd). Concentration determinationexperiments are also feasible.

The formulation of the present invention exhibits almost no loss inbiological activities of the polypeptide during the prolonged storageunder the conditions described above, as assessed by variousimmunological assays including, for example, enzyme-linked immunosorbentassay (ELISA) and Surface Plasmon Resonance to measure the ability ofthe polypeptide to specifically bind to an antigen. The polypeptidespresent in the formulation of the present invention retain, even underthe above defined stress conditions (such as storage under certaintemperature stress for defined periods) more than 80%, more than 85%,more than 90%, more than 95%, more than 98%, more than 99%, or more than99.5% of their initial biological activities (e.g., the ability to bindto RANKL, IL-6R, IL-23 and/or HSA) of the polypeptides prior to thestorage. In some embodiments, the polypeptides in the formulation of theinvention retain under the above defined stress conditions at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, orat least 99.5% of the biological activity (e.g., the ability to bind toRANKL, IL-6R, IL-23 and/or HSA) compared to the polypeptides present ina reference formulation prior to the storage.

In one embodiment, the polypeptides of the invention binds HSA. In theformulations of the present invention, at least 80% (preferably at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least99.5%) of said polypeptides retain their binding activity to HSA underone or more of the above stress conditions (such as storage undercertain temperature stress for defined periods) compared to the bindingactivity prior to the stress condition. Without being limiting, thebinding of the polypeptides to HSA can be determined e.g. by ELISAand/or BIACORE.

In another embodiment, the polypeptides of the invention bind RANKL. Inthe formulation of the present invention at least 80% (at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)of said polypeptides retain their binding activity to RANKL afterstorage under one or more of the above stress conditions compared to thebinding activity prior to storage.

In another embodiment, the polypeptides of the invention bind IL-6R. Inthe formulation of the present invention at least 80% (at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)of said polypeptides retain their binding activity to IL-6R afterstorage under one or more of the above stress conditions compared to thebinding activity prior to storage.

In another embodiment, the polypeptides of the invention bind IL-23. Inthe formulation of the present invention at least 80% (at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)of said polypeptides retain their binding activity to IL-23 afterstorage under one or more of the above stress conditions compared to thebinding activity prior to storage.

In a preferred aspect, at least 80% (at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5%) of thepolypeptides present in the formulation of the invention retain theirbinding activity to all of their targets (such as e.g. RANKL and HSA,IL-6R and HSA or IL-23 and HSA) after storage under one or more of theabove stress conditions compared to the binding activity prior tostorage.

Suitable animal models for determining the potency and/or biologicalactivity of the polypeptides present in the formulations of theinvention will be clear to the skilled person and will depend on theintended disease and/or disorder to be prevented and/or treated by thepolypeptide of the invention. Suitable animal models for testing thepotency and/or biological activity of SEQ ID NO's: 1 to 6 are e.g.described in WO 08/020079, WO 09/068627 and WO 08/142164.

Little to no loss of potency of the polypeptides of the invention hasbeen observed in formulations with a histidine buffer and informulations that comprise an excipient, preferably a saccharide,non-reducing sugar and/or polyol such as mannitol, sorbitol, trehaloseor sucrose. Accordingly, the present invention relates to a formulationcomprising an aqueous carrier and a polypeptide comprising one or moresingle variable domains, said formulation being formulated foradministration to a human subject, wherein said formulation furthercomprises at least one of:

-   -   a) A histidine buffer at a concentration of 10 mM to 100 mM        (preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as        15 mM or 10 mM);    -   b) An excipient, preferably a saccharide, non-reducing sugar        and/or polyol such as mannitol, sorbitol, trehalose or sucrose        at a concentration of 1% to 20% (preferably 2.5% to 15%, more        preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%),        wherein said formulation has an inorganic salt concentration of        150 mM or lower; and        wherein at least 80% (preferably at least 85%, at least 90%, at        least 95%, at least 98%, at least 99%, or at least 99.5%) of the        polypeptides retain their binding activity to at least one        (preferably to all) of their targets under one or more of the        above stress conditions (such as during storage at a temperature        of 37±5° C. up to at least 2 weeks (preferably at least 3 weeks,        at least 5 weeks, at least 8 weeks, at least 10 weeks, at least        3 months, at least 6 months, at least 1 year, 1.5 year or even 2        years or more)) compared to the binding activity prior to the        stress conditions, said binding activity as measured by ELISA        and/or BIACORE. In a preferred aspect, the formulation comprises        a histidine buffer at a concentration of 10 mM to 100 mM        (preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as        15 mM or 10 mM) and an excipient, preferably a saccharide,        non-reducing sugar and/or polyol such as mannitol, sorbitol,        trehalose or sucrose at a concentration of 1% to 20% (preferably        2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or        10%), such as e.g. 15 mM histidine pH 6.5 and 8% sucrose; or 10        mM histidine pH 6.0 and 10% sucrose.

Accordingly, in the stable formulations of the present inventionpreferably:

-   -   the polypeptide of the invention has a solubility of at least 20        mg/mL, at least 50 mg/mL, preferably at least 90 mg/mL, at least        120 mg/mL, at least 150 mg/mL or even 200 mg/mL or more) (e.g.        as assessed by PEG exclusion method or by centrifugal        ultrafiltration);    -   the polypeptide of the invention has a melting temperature of at        least 59° C. or more (such as 59.5° C. or more), preferably at        least 60° C. or more (such as 60.5° C. or more), more preferably        at least 61° C. or more (such as 61.5° C. or more) or at least        62° C. or more (such as 62.5° C. or more), most preferably at        least 63° C. or more (such as 63.5° C. or more) (e.g. as        assessed by TSA or DSC);    -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms pyroglutamate at the        N-terminal glutamic acid (e.g. as assessed by RP-HPLC) during        storage under one or more (of the above) stress conditions, such        as e.g. at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more);    -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms dimers (e.g. as assessed by        SE-HPLC) during storage under one or more (of the above) stress        conditions, such as e.g. at a temperature of 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 8 weeks, at least 10 weeks, at least 3 months, at least 6        months, at least 1 year, 1.5 year or even 2 years or more);    -   at least 80% (at least 85%, at least 90%, at least 95%, at least        98%, at least 99%, or at least 99.5%) of the polypeptides of the        invention retain their binding activity (e.g. as assessed by        ELISA and/or BIACORE) to at least one (preferably to all) of        their targets after storage under one or more (of the above)        stress conditions, such as e.g. at a temperature of 37±5° C. up        to at least 2 weeks (preferably at least 3 weeks, at least 5        weeks, at least 8 weeks, at least 10 weeks, at least 3 months,        at least 6 months, at least 1 year, 1.5 year or even 2 years or        more) compared to the binding activity prior to the stress        condition; and/or    -   the polypeptide of the invention is stable under one or more of        the following mechanical stress conditions:        -   shaking the formulation during 10 s to 1 min;        -   pushing the formulation through a needle (25G, preferably            26G, more preferably 27G, even more preferably 28G, most            preferably 29G or more) with a syringe (the syringe used can            be any commercially available syringe, such as e.g. a 1 mL,            2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL up to 50            mL syringe);        -   rotating for two days at 10 rpm; and/or        -   stirring for 1 hour at room temperature and/or 4-48 hours at            4° C. at at least 10 rpm (such as 50 rpm, 100 rpm or more).

An example of a preferred formulation of the invention with thesecharacteristics comprises 10 mg/mL of the polypeptide of the invention,15 mM histidine pH 6.5, 8% sucrose and 0.01% TWEEN (polysorbate) 80.Another example of a preferred formulation of the invention with thesecharacteristics comprises 10 mg/mL of the polypeptide of the invention,10 mM histidine pH 6.0, 10% sucrose and 0.005% TWEEN (polysorbate) 80.

General methods for producing the single variable domains and/orpolypeptides present in the formulation of the invention are known tothe skilled person and/or have been described in the art. The singlevariable domains and/or polypeptides can be produced in any host knownto the skilled person. For example but without being limiting, thesingle variable domains and/or polypeptides can be produced inprokaryotic hosts among which E. coli or eukaryotic hosts, for exampleeukaryotic host selected from insect cells, mammalian cells, and lowereukaryotic hosts comprising yeasts such as Pichia, Hansenula,Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora,Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, preferably Pichia pastoris. Production ofNanobodies in prokaryotes and lower eukaryotic hosts such as Pichiapastoris has e.g. been described in WO 94/04678, WO 94/25591 and WO08/142164. The contents of these applications are explicitly referred toin the connection with general culturing techniques and methods,including suitable media and conditions. The contents of these documentsare incorporated by reference. The skilled person can also devisesuitable genetic constructs for expression of the polypeptides of theinvention in different hosts on the basis of the present application andcommon general knowledge. The present invention also relates toconditions and genetic constructs described in the art, for example thegeneral culturing methods, plasmids, promoters and leader sequencesdescribed in WO 94/25591, WO 08/020079, Gasser et al. 2006 (Biotechnol.Bioeng. 94: 535); Gasser et al. 2007 (Appl. Environ. Microbiol. 73:6499); or Damasceno et al. 2007 (Microbiol. Biotechnol. 74: 381).

More particularly, the method for the expression and/or production of apolypeptide comprising one or more single variable domains at leastcomprising the steps of:

-   -   a) cultivating a host or host cell (as defined herein) under        conditions that are such that said host or host cell will        multiply;    -   b) maintaining said host or host cell under conditions that are        such that said host or host cell expresses and/or produces the        polypeptide;    -   c) isolating and/or purifying the secreted polypeptide from the        medium.

To produce/obtain expression of the polypeptide, the transformed hostcell or transformed host organism may generally be kept, maintainedand/or cultured under conditions such that the (desired) polypeptide isexpressed/produced. Suitable conditions will be clear to the skilledperson and will usually depend upon the host cell/host organism used, aswell as on the regulatory elements that control the expression of the(relevant) nucleotide sequence. Again, reference is made to thehandbooks and patent applications mentioned above.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

The polypeptide of the invention may then be isolated from the hostcell/host organism and/or from the medium in which said host cell orhost organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the polypeptide of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the polypeptide to be isolated).

In the present invention, the host can be removed from the culturemedium by routine means. For example, the host can be removed bycentrifugation or filtration. The solution obtained by removal of thehost from the culture medium is also referred to as culture supernatant,or clarified culture supernatant. The polypeptides of the invention canbe purified from the culture supernatant by standard methods. Standardmethods include, but are not limited to chromatographic methods,including size exclusion chromatography, hydrophobic interactionchromatography, ion exchange chromatography, and affinitychromatography. These methods can be performed alone or in combinationwith other purification methods, e.g. precipitation or gelelectrophoresis. The skilled person can devise suitable combinations ofpurification methods for the polypeptides of the invention on the basisof common general knowledge. For specific examples the art cited hereinis referred to.

In one exemplary embodiment, the polypeptides of the invention can bepurified from culture supernatant by a combination of affinitychromatography on Protein A, ion exchange chromatography and sizeexclusion chromatography. Reference to any “step of purification”,includes, but is not limited to these particular methods.

More specifically, the polypeptides of the invention can be purifiedfrom culture supernatant using a process wherein the clarifiedsupernatant (obtained by centrifugation) is captured on any combinationof columns selected from (without being limiting) affinitychromatography resin such as Protein A resin, Cation ExchangeChromatography (CIEC) or an Anion Exchange Chromatography (AIEC) usingfor example Poros 50HS (POROS), SOURCE 30S or SOURCE 15S (GEHealthcare), SP SEPHAROSE (GE Healthcare), CAPTO S (GE Healthcare),CAPTO MMC (GE Healthcare) or Poros 50HQ (POROS), SOURCE 30Q or SOURCE15Q (GE Healthcare), Q SEPHAROSE (GE Healthcare), CAPTO Q and DEAESEPHAROSE (GE Healthcare), Size exclusion chromatography (SE-HPLC) usingfor example SUPERDEX 75 or SUPERDEX 200 (GE Healthcare), hydrophobicinteraction chromatography (HIC) using for example octyl, butylSEPHAROSE or equivalents, optionally also including a tangential flowfiltration (TFF) step. Any combination of columns can be used for thepurification of the polypeptides of the invention, such as e.g. ProteinA resin followed by Cation Exchange Chromatography or two CationExchange Chromatography steps.

The present invention also provides methods for preparing the stableformulations of the invention comprising the polypeptides of theinvention. More particularly, the present invention provides methods forpreparing stable formulations of such polypeptides, said methodscomprising concentrating a fraction containing the purified polypeptideto the final polypeptide concentration using e.g. a semipermeablemembrane with an appropriate molecular weight (MW) cutoff (e.g. a 5 kDcutoff for single variable domains; a 10 kD cutoff for bivalentpolypeptides comprising two single variable domains; or a 15 kD cutofffor trivalent polypeptides comprising three single variable domains) anddiafiltering and/or ultrafiltering to buffer exchange and furtherconcentrate the polypeptide fraction into the formulation buffer usingthe same membrane. As extensively described above, the formulationbuffer of the present invention may further comprise at least one of:

-   -   a) A buffer at a concentration of 10 mM to 100 mM selected from        the group consisting of hepes pH 7.0-8.0, histidine pH 6.0-6.5,        MES pH 6.0 and acetate pH 5.5-6.0;    -   b) An excipient at a concentration of 1% to 20%;    -   c) A surfactant at a concentration of 0.001% to 1% selected from        TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.

The pH of the formulation may range from about 5.5 to about 8.0, or mayrange from about 6.0 to about 7.5, preferably from about 6.2 to 7.5,from about 6.2 to 7.0, most preferably from about 6.5 to 7.0.

Surfactant (e.g. TWEEN (polysorbate) 20, TWEEN (polysorbate) 80 orpoloxamer) will be added after the final diafiltration/ultrafiltrationstep at a concentration in the range of about 0% to 1%, preferably0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%,0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or0.005%.

The formulation of the present invention may be sterilized by varioussterilization methods, including sterile filtration, radiation, etc. Ina specific embodiment, the polypeptide formulation is filter-sterilizedwith a presterilized 0.2 micron filter.

Preferably, the formulation of the present invention is supplied in ahermetically sealed container. Liquid formulations may comprise aquantity between 1 mL and 20 mL, preferably about 1 mL, 2 mL, 3 mL, 4mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20 mL.

The formulation of the present invention can be prepared as unit dosageforms by preparing a vial containing an aliquot of the formulation for aone time use. For example, a unit dosage of liquid formulation per vialmay contain 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL,15 mL, or 20 mL of the formulation. The pharmaceutical unit dosage formscan be made suitable for any form of delivery of the polypeptide of theinvention including (without being limiting) parenteral delivery,topical delivery, pulmonary delivery, intranasal delivery, vaginaldelivery, enteral delivery, rectal delivery, oral delivery and/orsublingual delivery. In one aspect, the present invention relates to apharmaceutical unit dosage form suitable for parenteral (such as e.g.intravenous, intraarterial, intramuscular, intracerebral, intraosseous,intradermal, intrathecal, intraperitoneal, subcutaneous, etc.)administration to a subject, comprising a formulation of the inventionin a suitable container. In another preferred aspect, the subject is ahuman. In a specific embodiment, the formulations of the presentinvention are formulated into single dose vials as a sterile liquid thatcontains 10 mg/mL of one of SEQ ID NO's: 1 to 6, 15 mM histidine bufferat pH 6.5, 8% sucrose and 0.01% TWEEN (polysorbate) 80. In anotherspecific embodiment, the formulations of the present invention areformulated into single dose vials as a sterile liquid that contains 10mg/mL of one of SEQ ID NO's: 1 to 6, 10 mM histidine buffer at pH 6.0,10% sucrose and 0.005% TWEEN (polysorbate) 80.

The amount of a formulation of the present invention which will beeffective in the prevention, treatment and/or management of a certaindisease or disorder can be determined by standard clinical techniqueswell-known in the art or described herein. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. For formulations of the polypeptide, encompassed bythe invention, the dosage administered to a patient may further becalculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg.

The required volume (in mL) to be given is then determined by taking themg dose required divided by the concentration of the polypeptideformulation. The final calculated required volume will be obtained bypooling the contents of as many vials as are necessary into syringe(s)to administer the polypeptide formulation of the invention.

The present invention also encompasses a finished packaged and labelledpharmaceutical product. This article of manufacture or kit includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In oneembodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses formulations, preferably sterile, suitable foreach delivery route. In the case of dosage forms suitable for parenteraladministration (such as e.g. subcutaneous administration) the activeingredient, e.g., polypeptide of the invention, is sterile and suitablefor administration as a particulate free solution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises the formulation containing the polypeptide. Thepackaging material includes instruction means which indicate that saidpolypeptide can be used to prevent, treat and/or manage one or moresymptoms associated with the disease or disorder by administeringspecific doses and using specific dosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a formulation containing the polypeptide of interest, andwherein said packaging material includes instruction means whichindicate that said agents can be used to prevent, treat and/or managethe disease or disorder by administering specific doses and usingspecific dosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a formulation containing the polypeptide, and wherein saidpackaging material includes instruction means which indicate that saidagents can be used to prevent, treat and/or manage one or more symptomsassociated with the disease or disorder by administering specific dosesand using specific dosing regimens as described herein.

The formulations, containers, pharmaceutical unit dosages and kits ofthe present invention may be administered to a subject to prevent, treatand/or manage a specific disease and/or disorder. In a specific aspect,the formulations, containers, pharmaceutical unit dosages and kits ofthe present invention are administered to a subject to prevent, treatand/or manage a disease and/or disorder associated with or characterizedby aberrant expression and/or activity of a certain target or one ormore symptoms thereof. In another specific aspect, the formulations,containers, pharmaceutical unit dosages and kits of the presentinvention are administered to a subject to prevent, treat and/or managediseases and/or disorders associated with aberrant expression and/oractivity of RANKL, diseases and/or disorders associated withoverexpression of IL-6, or diseases and disorders associated withheterodimeric cytokines and their receptors or one or more symptomsthereof.

Diseases and disorders associated with aberrant expression and/oractivity of RANKL are for example bone diseases and disorders, andinclude (without being limiting) the following diseases and disorders:Osteoporosis (McClung 2006, Current Osteoporosis Reports 4: 28-33),including, but not limited to, primary osteoporosis, endocrineosteoporosis (including, but not limited to, hyperthyroidism,hyperparathyroidism (Anandarajah and Schwarz 2006, J. Cell Biochem. 97:226-232), Cushing's syndrome, and acromegaly), hereditary and congenitalforms of osteoporosis (including, but not limited to, osteogenesisimperfecta, homocystinuria, Menkes' syndrome, Riley-Day syndrome),osteoporosis due to immobilization of extremities,glucocorticoid-induced osteoporosis (Locklin et al. 2001, Bone 28(Suppl.): 580; McClung 2006, Current Osteoporosis Reports 4: 28-33;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232) andpost-menopausal osteoporosis (McClung 2006, Current Osteoporosis Reports4: 28-33); (Juvenile or Familial) Paget's disease (Cundy et al. 2002,Hum. Mol. Genet. 11: 2119-2127; Whyte et al. 2002, J. Bone Miner. Res.17: 26-29; Whyte et al. 2002, N. Engl. J. Med. 347: 175-184;Johnson-Pais et al. 2003, J. Bone Miner Res. 18: 376-380; Anandarajahand Schwarz 2006, J. Cell Biochem. 97: 226-232; Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232); Osteomyelitis, i.e., an infectiouslesion in bone, leading to bone loss; Hypercalcemia (Anandarajah andSchwarz 2006, J. Cell Biochem. 97: 226-232), including, but not limitedto, hypercalcemia resulting from solid tumors (including, but notlimited to, breast, lung and kidney) and hematologic malignancies(including, but not limited to, multiple myeloma (Sordillo and Pearse2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003, CancerRes. 63: 287-289), lymphoma and leukemia), idiopathic hypercalcemia, andhypercalcemia associated with hyperthyroidism and renal functiondisorders; Bone loss, including but not limited to, osteopenia followingsurgery, osteopenia induced by steroid administration, osteopeniaassociated with disorders of the small and large intestine, andosteopenia associated with chronic hepatic and renal diseases;Osteonecrosis, i.e., bone cell death, including, but not limited to,osteonecrosis associated with traumatic injury, osteonecrosis associatedwith Gaucher's disease, osteonecrosis associated with sickle cellanemia, osteonecrosis associated with systemic lupus erythematosus,osteonecrosis associated with rheumatoid arthritis, osteonecrosisassociated with periodontal disease, osteonecrosis associated withosteolytic metastasis, and osteonecrosis associated with othercondition; Bone loss associated with arthritic disorders such aspsoriatic arthritis, rheumatoid arthritis, loss of cartilage and jointerosion associated with rheumatoid arthritis (Bezerra et al. 2005,Brazilian Journal of Medical and Biological Research 38: 161-170;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170), including inflammatory arthritis (McClung 2006,Current Osteoporosis Reports 4: 28-33), Collagen-induced arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170); Periprosthetic osteolysis (McClung 2006, CurrentOsteoporosis Reports 4: 28-33; Anandarajah and Schwarz 2006, J. CellBiochem. 97: 226-232); Cancer-related bone disease (McClung 2006,Current Osteoporosis Reports 4: 28-33); Bone loss associated witharomatase inhibitor therapy (Lewiecki 2006, Expert Opin. Biol. Ther. 6:1041-1050); Bone loss associated with androgen deprivation therapy(Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050); Bone lossassociated bone metastasis; Bone loss associated with diseases havingimmune system involvement, such as adult and childhood leukaemias,cancer metastasis, autoimmunity, and various viral infections (HolsteadJones et al. 2002, Ann. Rheum. Dis. 61 (Suppl II): ii32-ii39);Osteopenic disorders such as adult and childhood leukaemia (Oliveri etal. 1999, Henry Ford Hosp. Med. 39: 45-48); chronic infections such ashepatitis C or HIV (Stellon et al. 1985, Gastroenterology 89:1078-1083); autoimmune disorders such as diabetes mellitus (Piepkorn etal. 1997, Horm. Metab. Res. 29: 584-91), and lupus erythematosus (Seitzet al. 1985, Ann. Rheum Dis. 44: 438-445); allergic diseases such asasthma (Ebeling et al. 1998, J. Bone Min. Res. 13: 1283-1289); lyticbone metastases in multiple cancers such as breast cancer (Coleman 1998,Curr. Opin. Oncol. 10 (Suppl 1): 7-13); Prostate cancer; Myeloma bonedisease (Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232);Periodontal infections (Anandarajah and Schwarz 2006, J. Cell Biochem.97: 226-232); Expansile skeletal hyperphosphatasia (Anandarajah andSchwarz 2006, J. Cell Biochem. 97: 226-232); Bone metastases (Lewiecki2006, Expert Opin. Biol. Ther. 6: 1041-1050; Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the formulations, containers,pharmaceutical unit dosages and kits of the invention of other diseasesand disorders associated with an imbalance in the RANKL/RANK/OPGpathway. Such diseases and disorders include but are not limited toosteoporosis, inflammatory conditions, autoimmune conditions, asthma,rheumatoid arthritis, multiple sclerosis, Multiple myeloma (Sordillo andPearse 2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003,Cancer Res. 63: 287-289); Vascular diseases (Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232) and Cardiovascular disease (Lewiecki2006, Expert Opin. Biol. Ther. 6: 1041-1050).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the formulations, containers,pharmaceutical unit dosages and kits of the invention of diseases anddisorders associated with osteopetrosis such as osteopetrosis tarda,osteopetrosis congenita and marble bone disease.

Disease and disorders caused by aberrant expression and or activity,such as excessive IL-6 production or signaling include sepsis (Starneset al., 1999) and various forms of cancer such as multiple myelomadisease (MM), renal cell carcinoma (RCC), plasma cell leukaemia (Kleinet al., 1991), lymphoma, B-lymphoproliferative disorder (BLPD) andprostate cancer. Non-limiting examples of other diseases caused byaberrant expression and/or activity, such as excessive IL-6 productionor signalling include bone resorption (osteoporosis) (Roodman et al.,1992; Jilka et al., 1992), cachexia (Strassman et al., 1992), psoriasis,mesangial proliferative glomerulonephritis, Kaposi's sarcoma,AIDS-related lymphoma (Emilie et al., 1994), inflammatory diseases anddisorder such as rheumatoid arthritis, systemic onset juvenileidiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990),Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE),multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma,asthma (in particular allergic asthma) and autoimmune insulin-dependentdiabetes mellitus (Campbell et al., 1991).

Diseases and disorders associated with heterodimeric cytokines and theirreceptors include inflammation and inflammatory disorders such as boweldiseases (colitis, Crohn's disease, IBD), infectious diseases,psoriasis, cancer, autoimmune diseases (such as MS), carcoidis,transplant rejection, cystic fibrosis, asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, viral infection, commonvariable immunodeficiency.

The formulations, containers, pharmaceutical unit dosages and kits ofthe present invention may also be advantageously utilized in combinationwith one or more other therapies (e.g., one or more other prophylacticor therapeutic agents), preferably therapies useful in the prevention,treatment and/or management of the (same or another) disease ordisorder. When one or more other therapies (e.g., prophylactic ortherapeutic agents) are used, they can be administered separately, inany appropriate form and by any suitable route. Therapeutic orprophylactic agents include, but are not limited to, small molecules,synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g.,DNA and RNA nucleotides including, but not limited to, antisensenucleotide sequences, triple helices, RNAi, and nucleotide sequencesencoding biologically active proteins, polypeptides or peptides),antibodies, other single variable domains, synthetic or naturalinorganic molecules, mimetic agents, and synthetic or natural organicmolecules. Any therapy (e.g., prophylactic or therapeutic agents) whichis known to be useful, or which has been used or is currently being usedfor the prevention, treatment and/or management of one or more symptomsassociated with a specific disease or disorder, can be used incombination with the formulations of the present invention in accordancewith the invention described herein.

A formulation of the invention may be administered to a mammal,preferably a human, concurrently with one or more other therapies (e.g.,one or more other prophylactic or therapeutic agents). The term“concurrently” is not limited to the administration of prophylactic ortherapeutic agents/therapies at exactly the same time, but rather it ismeant that the formulation of the invention and the other agent/therapyare administered to a mammal in a sequence and within a time intervalsuch that the polypeptide contained in the formulation can act togetherwith the other agent/therapy to provide an increased benefit than ifthey were administered otherwise. For example, the formulation of theinvention and the one or more other prophylactic or therapeutic agentsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic or prophylactic effect.

When used in combination with other therapies (e.g., prophylactic and/ortherapeutic agents), the formulations of the invention and the othertherapy can act additively or synergistically. The inventioncontemplates administration of a formulation of the invention incombination with other therapies (e.g., prophylactic or therapeuticagents) by the same or different routes of administration, e.g., oraland parenteral.

Various delivery systems are known and can be used to administer theformulation of the present invention. Methods of administeringformulations of the present invention include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and, preferably subcutaneous), epiduraladministration, topical administration, and mucosal administration(e.g., intranasal and oral routes). In a specific embodiment, liquidformulations of the present invention are administered parenteral.

DEFINITIONS

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10^(th) Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein;

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein; as well as to for example thefollowing reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56;Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J.Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000,21 Suppl. A, 5106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43,which describe techniques for protein engineering, such as affinitymaturation and other techniques for improving the specificity and otherdesired properties of proteins such as immunoglobulins.

Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as mentioned in Table A-2.

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0- Leucine Leu L 7.0)⁽³⁾Isoleucine Ile I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan TrpW Proline Pro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH6.0-7.0) Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln QTyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH6.0-7.0) Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes:⁽¹⁾Sometimes also considered to be a polar uncharged amino acid.⁽²⁾Sometimes also considered to be a nonpolar uncharged amino acid.⁽³⁾As will be clear to the skilled person, the fact that an amino acidresidue is referred to in this Table as being either charged oruncharged at pH 6.0 to 7.0 does not reflect in any way on the chargesaid amino acid residue may have at a pH lower than 6.0 and/or at a pHhigher than 7.0; the amino acid residues mentioned in the Table can beeither charged and/or uncharged at such a higher or lower pH, as will beclear to the skilled person. ⁽⁴⁾As is known in the art, the charge of aHis residue is greatly dependant upon even small shifts in pH, but a Hisresidu can generally be considered essentially uncharged at a pH ofabout 6.5.

For the purposes of comparing two or more nucleotide sequences, thepercentage of “sequence identity” between a first nucleotide sequenceand a second nucleotide sequence may be calculated by dividing [thenumber of nucleotides in the first nucleotide sequence that areidentical to the nucleotides at the corresponding positions in thesecond nucleotide sequence] by [the total number of nucleotides in thefirst nucleotide sequence] and multiplying by [100%], in which eachdeletion, insertion, substitution or addition of a nucleotide in thesecond nucleotide sequence—compared to the first nucleotide sequence—isconsidered as a difference at a single nucleotide (position).

Alternatively, the degree of sequence identity between two or morenucleotide sequences may be calculated using a known computer algorithmfor sequence alignment such as NCBI Blast v2.0, using standard settings.

-   -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO        00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence.

For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence (also referred to herein as “amino acididentity”) may be calculated by dividing [the number of amino acidresidues in the first amino acid sequence that are identical to theamino acid residues at the corresponding positions in the second aminoacid sequence] by [the total number of amino acid residues in the firstamino acid sequence] and multiplying by [100%], in which each deletion,insertion, substitution or addition of an amino acid residue in thesecond amino acid sequence—compared to the first amino acid sequence—isconsidered as a difference at a single amino acid residue (position),i.e. as an “amino acid difference” as defined herein.

Alternatively, the degree of sequence identity between two amino acidsequences may be calculated using a known computer algorithm, such asthose mentioned above for determining the degree of sequence identityfor nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two amino acid sequences in accordance with thecalculation method outlined hereinabove, the amino acid sequence withthe greatest number of amino acid residues will be taken as the “first”amino acid sequence, and the other amino acid sequence will be taken asthe “second” amino acid sequence.

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 andWO 01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferredconservative substitutions are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln;Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, intoGln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Natl. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,1986, all incorporated herein in their entirety by reference.Information on the primary, secondary and tertiary structure ofNanobodies® is given in the description herein and in the generalbackground art cited above. Also, for this purpose, the crystalstructure of a V_(HH) domain from a llama is for example given byDesmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);Spinelli et al., Natural Structural Biology (1996); 3, 752-757; andDecanniere et al., Structure, Vol. 7, 4, 361 (1999). Further informationabout some of the amino acid residues that in conventional V_(H) domainsform the V_(H)/V_(L) interface and potential camelizing substitutions onthese positions can be found in the prior art cited above.

Amino acid sequences and nucleic acid sequences are said to be “exactlythe same” if they have 100% sequence identity (as defined herein) overtheir entire length.

When comparing two amino acid sequences, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first sequence, compared to thesecond sequence; it being understood that two amino acid sequences cancontain one, two or more such amino acid differences.

When a nucleotide sequence or amino acid sequence is said to “comprise”another nucleotide sequence or amino acid sequence, respectively, or to“essentially consist of” another nucleotide sequence or amino acidsequence, this may mean that the latter nucleotide sequence or aminoacid sequence has been incorporated into the first mentioned nucleotidesequence or amino acid sequence, respectively, but more usually thisgenerally means that the first mentioned nucleotide sequence or aminoacid sequence comprises within its sequence a stretch of nucleotides oramino acid residues, respectively, that has the same nucleotide sequenceor amino acid sequence, respectively, as the latter sequence,irrespective of how the first mentioned sequence has actually beengenerated or obtained (which may for example be by any suitable methoddescribed herein). By means of a non-limiting example, when a Nanobody®of the invention is said to comprise a CDR sequence, this may mean thatsaid CDR sequence has been incorporated into the Nanobody® of theinvention, but more usually this generally means that the Nanobody® ofthe invention contains within its sequence a stretch of amino acidresidues with the same amino acid sequence as said CDR sequence,irrespective of how said Nanobody® of the invention has been generatedor obtained. It should also be noted that when the latter amino acidsequence has a specific biological or structural function, it preferablyhas essentially the same, a similar or an equivalent biological orstructural function in the first mentioned amino acid sequence (in otherwords, the first mentioned amino acid sequence is preferably such thatthe latter sequence is capable of performing essentially the same, asimilar or an equivalent biological or structural function). Forexample, when a Nanobody® of the invention is said to comprise a CDRsequence or framework sequence, respectively, the CDR sequence andframework are preferably capable, in said Nanobody®, of functioning as aCDR sequence or framework sequence, respectively. Also, when anucleotide sequence is said to comprise another nucleotide sequence, thefirst mentioned nucleotide sequence is preferably such that, when it isexpressed into an expression product (e.g. a polypeptide), the aminoacid sequence encoded by the latter nucleotide sequence forms part ofsaid expression product (in other words, that the latter nucleotidesequence is in the same reading frame as the first mentioned, largernucleotide sequence).

A nucleic acid sequence or amino acid sequence is considered to be “(in)essentially isolated (form)”—for example, compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another nucleic acid, another protein/polypeptide,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a nucleic acidsequence or amino acid sequence is considered “essentially isolated”when it has been purified at least 2-fold, in particular at least10-fold, more in particular at least 100-fold, and up to 1000-fold ormore. A nucleic acid sequence or amino acid sequence that is “inessentially isolated form” is preferably essentially homogeneous, asdetermined using a suitable technique, such as a suitablechromatographical technique, such as polyacrylamide-gel electrophoresis.

The term “domain” as used herein generally refers to a globular regionof an amino acid sequence (such as an antibody chain, and in particularto a globular region of a heavy chain antibody), or to a polypeptidethat essentially consists of such a globular region. Usually, such adomain will comprise peptide loops (for example 3 or 4 peptide loops)stabilized, for example, as a sheet or by disulfide bonds. The term“binding domain” refers to such a domain that is directed against anantigenic determinant (as defined herein).

The term “antigenic determinant” refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as a Nanobody® or apolypeptide of the invention) and more in particular by theantigen-binding site of said molecule. The terms “antigenic determinant”and “epitope” may also be used interchangeably herein.

As further described in paragraph m) on page 53 of WO 08/020079, anamino acid sequence (such as a Nanobody, an antibody, a polypeptide ofthe invention, or generally an antigen binding protein or polypeptide ora fragment thereof) that can (specifically) bind to, that has affinityfor and/or that has specificity for a specific antigenic determinant,epitope, antigen or protein (or for at least one part, fragment orepitope thereof) is said to be “against” or “directed against” saidantigenic determinant, epitope, antigen or protein.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as a Nanobody® or apolypeptide of the invention) molecule can bind. The specificity of anantigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (K_(D)), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)). As will be clear to the skilled person (for exampleon the basis of the further disclosure herein), affinity can bedetermined in a manner known per se, depending on the specific antigenof interest. Avidity is the measure of the strength of binding betweenan antigen-binding molecule (such as a Nanobody® or polypeptide of theinvention) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule. Typically,antigen-binding proteins (such as the amino acid sequences, Nanobodies®and/or polypeptides of the invention) will bind to their antigen with adissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, andpreferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or moreand more preferably 10⁸ to 10¹² liter/moles). Any K, value greater than10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹) liters/mol isgenerally considered to indicate non-specific binding. Preferably, amonovalent immunoglobulin sequence of the invention will bind to thedesired antigen with an affinity less than 500 nM, preferably less than200 nM, more preferably less than 10 nM, such as less than 500 pM.Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variantsthereof known per se in the art; as well as the other techniquesmentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more then 10⁻⁴ moles/liter or 10⁻³ moles/liter(e.g. of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(K_(A)), by means of the relationship [K_(D)=1/K_(A)].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the K_(D), ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, K_(A), which equals1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two molecules(such as an amino acid sequence, Nanobody® or polypeptide of theinvention and its intended target) will mainly be expressed in terms ofthe K_(D) value of their interaction; it being clear to the skilledperson that in view of the relation K_(A)=1/K_(D), specifying thestrength of molecular interaction by its K_(D) value can also be used tocalculate the corresponding K_(A) value. The K_(D)-value characterizesthe strength of a molecular interaction also in a thermodynamic sense asit is related to the free energy (DG) of binding by the well knownrelation DG=RT·ln(K_(D)) (equivalently DG=−RT·ln(K_(A))), where R equalsthe gas constant, T equals the absolute temperature and ln denotes thenatural logarithm. The K_(D) for biological interactions which areconsidered meaningful (e.g. specific) are typically in the range of10⁻¹⁰M (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is, thelower is its K_(D). The K_(D) can also be expressed as the ratio of thedissociation rate constant of a complex, denoted as k_(off), to the rateof its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe SI unit notation of second). The on-rate k_(on) has units M⁻¹s⁻¹.The on-rate may vary between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, approachingthe diffusion-limited association rate constant for bimolecularinteractions. The off-rate is related to the half-life of a givenmolecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate may vary between 10⁻⁶ s⁻¹ (near irreversible complex with at_(1/2) of multiple days) to 1 s⁻¹ t_(1/2)=0.69 s).

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern. Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(on),k_(off) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artefacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition sites for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of K_(D) may be quite labor-intensiveand as consequence, often apparent K_(D) values are determined to assessthe binding strength of two molecules. It should be noted that as longall measurements are made in a consistent way (e.g. keeping the assayconditions unchanged) apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or FACS (Fluorescent activatedcell sorting) or other format (the fluorophore for fluorescencedetection, the chromophore for light absorption detection, the biotinfor streptavidin-mediated ELISA detection). Typically, the referencemolecule C is kept at a fixed concentration and the concentration of Ais varied for a given concentration or amount of B. As a result an IC₅₀value is obtained corresponding to the concentration of A at which thesignal measured for C in absence of A is halved. Provided K_(D ref), theK_(D) of the reference molecule, is known, as well as the totalconcentration c_(ref) of the reference molecule, the apparent K_(D) forthe interaction A-B can be obtained from following formula:K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if c_(ref)<<K_(D ref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the strength or stability of a molecular interaction can beassessed by the IC₅₀ and this measurement is judged as equivalent toK_(D) or to apparent K_(D) throughout this text.

The half-life of an amino acid sequence, compound or polypeptide of theinvention can generally be defined as the time taken for the serumconcentration of the amino acid sequence, compound or polypeptide to bereduced by 50%, in vivo, for example due to degradation of the sequenceor compound and/or clearance or sequestration of the sequence orcompound by natural mechanisms. The in vivo half-life of an amino acidsequence, compound or polypeptide of the invention can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to awarm-blooded animal (i.e. to a human or to another suitable mammal, suchas a mouse, rabbit, rat, pig, dog or a primate, for example monkeys fromthe genus Macaca (such as, and in particular, cynomolgus monkeys (Macacafascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papioursinus)) a suitable dose of the amino acid sequence, compound orpolypeptide of the invention; collecting blood samples or other samplesfrom said animal; determining the level or concentration of the aminoacid sequence, compound or polypeptide of the invention in said bloodsample; and calculating, from (a plot of) the data thus obtained, thetime until the level or concentration of the amino acid sequence,compound or polypeptide of the invention has been reduced by 50%compared to the initial level upon dosing. Reference is for example madeto the Experimental Part below, as well as to the standard handbooks,such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and Peters et al, Pharmacokinete analysis: APractical Approach (1996). Reference is also made to “Pharmacokinetics”,M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition(1982).

As will also be clear to the skilled person (see for example pages 6 and7 of WO 04/003019 and in the further references cited therein), thehalf-life can be expressed using parameters such as the t½-alpha,t½-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, such as any two of these parameters, oressentially all three these parameters. As used herein “increase inhalf-life” or “increased half-life” in particular refers to an increasein the t½-beta, either with or without an increase in the t½-alphaand/or the AUC or both.

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, a target or antigen, asmeasured using a suitable in vitro, cellular or in vivo assay. Inparticular, “modulating” or “to modulate” may mean either reducing orinhibiting the activity of, or alternatively increasing a (relevant orintended) biological activity of, a target or antigen, as measured usinga suitable in vitro, cellular or in vivo assay (which will usuallydepend on the target or antigen involved), by at least 1%, preferably atleast 5%, such as at least 10% or at least 25%, for example by at least50%, at least 60%, at least 70%, at least 80%, or 90% or more, comparedto activity of the target or antigen in the same assay under the sameconditions but without the presence of the construct of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of a target or antigenfor one or more of its ligands, binding partners, partners forassociation into a homomultimeric or heteromultimeric form, orsubstrates; and/or effecting a change (which may either be an increaseor a decrease) in the sensitivity of the target or antigen for one ormore conditions in the medium or surroundings in which the target orantigen is present (such as pH, ion strength, the presence ofco-factors, etc.), compared to the same conditions but without thepresence of the construct of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, depending on the target orantigen involved.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist, as an antagonist or as a reverse agonist, respectively,depending on the target or antigen and the desired biological orphysiological effect) with respect to one or more biological orphysiological mechanisms, effects, responses, functions, pathways oractivities in which the target or antigen (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, depending on the target or antigeninvolved. In particular, an action as an agonist or antagonist may besuch that an intended biological or physiological activity is increasedor decreased, respectively, by at least 1%, preferably at least 5%, suchas at least 10% or at least 25%, for example by at least 50%, at least60%, at least 70%, at least 80%, or 90% or more, compared to thebiological or physiological activity in the same assay under the sameconditions but without the presence of the construct of the invention.

Modulating may for example also involve allosteric modulation of thetarget or antigen; and/or reducing or inhibiting the binding of thetarget or antigen to one of its substrates or ligands and/or competingwith a natural ligand, substrate for binding to the target or antigen.Modulating may also involve activating the target or antigen or themechanism or pathway in which it is involved. Modulating may for examplealso involve effecting a change in respect of the folding orconfirmation of the target or antigen, or in respect of the ability ofthe target or antigen to fold, to change its confirmation (for example,upon binding of a ligand), to associate with other (sub)units, or todisassociate. Modulating may for example also involve effecting a changein the ability of the target or antigen to transport other compounds orto serve as a channel for other compounds (such as ions).

Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

In respect of a target or antigen, the term “interaction site” on thetarget or antigen means a site, epitope, antigenic determinant, part,domain or stretch of amino acid residues on the target or antigen thatis a site for binding to a ligand, receptor or other binding partner, acatalytic site, a cleavage site, a site for allosteric interaction, asite involved in multi-merization (such as homomerization orheterodimerization) of the target or antigen; or any other site,epitope, antigenic determinant, part, domain or stretch of amino acidresidues on the target or antigen that is involved in a biologicalaction or mechanism of the target or antigen. More generally, an“interaction site” can be any site, epitope, antigenic determinant,part, domain or stretch of amino acid residues on the target or antigento which an amino acid sequence or polypeptide of the invention can bindsuch that the target or antigen (and/or any pathway, interaction,signalling, biological mechanism or biological effect in which thetarget or antigen is involved) is modulated (as defined herein).

An amino acid sequence or polypeptide is said to be “specific for” afirst target or antigen compared to a second target or antigen when isbinds to the first antigen with an affinity (as described above, andsuitably expressed as a K_(D) value, K_(A) value, K_(off) rate and/orK_(on) rate) that is at least 10 times, such as at least 100 times, andpreferably at least 1000 times, and up to 10,000 times or more betterthan the affinity with which said amino acid sequence or polypeptidebinds to the second target or polypeptide. For example, the firstantigen may bind to the target or antigen with a K_(D) value that is atleast 10 times less, such as at least 100 times less, and preferably atleast 1000 times less, such as 10,000 times less or even less than that,than the K_(D) with which said amino acid sequence or polypeptide bindsto the second target or polypeptide. Preferably, when an amino acidsequence or polypeptide is “specific for” a first target or antigencompared to a second target or antigen, it is directed against (asdefined herein) said first target or antigen, but not directed againstsaid second target or antigen.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an amino acid sequence orother binding agents (such as a polypeptide of the invention) tointerfere with the binding of other amino acid sequences or bindingagents of the invention to a given target. The extend to which an aminoacid sequence or other binding agents of the invention is able tointerfere with the binding of another to a target, and therefore whetherit can be said to cross-block according to the invention, can bedetermined using competition binding assays. One particularly suitablequantitative assay uses a BIACORE machine which can measure the extentof interactions using surface plasmon resonance technology. Anothersuitable quantitative cross-blocking assay uses an ELISA-based approachto measure competition between amino acid sequence or another bindingagents in terms of their binding to the target.

The following generally describes a suitable BIACORE assay fordetermining whether an amino acid sequence or other binding agentcross-blocks or is capable of cross-blocking according to the invention.It will be appreciated that the assay can be used with any of the aminoacid sequence or other binding agents described herein. The BIACOREmachine (for example the BIACORE 3000) is operated in line with themanufacturer's recommendations. Thus in one cross-blocking assay, thetarget protein is coupled to a CM5 BIACORE chip using standard aminecoupling chemistry to generate a surface that is coated with the target.Typically 200-800 resonance units of the target would be coupled to thechip (an amount that gives easily measurable levels of binding but thatis readily saturable by the concentrations of test reagent being used).Two test amino acid sequences (termed A* and B*) to be assessed fortheir ability to cross-block each other are mixed at a one to one molarratio of binding sites in a suitable buffer to create the test mixture.When calculating the concentrations on a binding site basis themolecular weight of an amino acid sequence is assumed to be the totalmolecular weight of the amino acid sequence divided by the number oftarget binding sites on that amino acid sequence. The concentration ofeach amino acid sequence in the test mix should be high enough toreadily saturate the binding sites for that amino acid sequence on thetarget molecules captured on the BIACORE chip. The amino acid sequencesin the mixture are at the same molar concentration (on a binding basis)and that concentration would typically be between 1.00 and 1.5micromolar (on a binding site basis). Separate solutions containing A*alone and B* alone are also prepared. A* and B* in these solutionsshould be in the same buffer and at the same concentration as in thetest mix. The test mixture is passed over the target-coated BIACORE chipand the total amount of binding recorded. The chip is then treated insuch a way as to remove the bound amino acid sequences without damagingthe chip-bound target. Typically this is done by treating the chip with30 mM HCl for 60 seconds. The solution of A* alone is then passed overthe target-coated surface and the amount of binding recorded. The chipis again treated to remove all of the bound amino acid sequences withoutdamaging the chip-bound target. The solution of B* alone is then passedover the target-coated surface and the amount of binding recorded. Themaximum theoretical binding of the mixture of A* and B* is nextcalculated, and is the sum of the binding of each amino acid sequencewhen passed over the target surface alone. If the actual recordedbinding of the mixture is less than this theoretical maximum then thetwo amino acid sequences are cross-blocking each other. Thus, ingeneral, a cross-blocking amino acid sequence or other binding agentaccording to the invention is one which will bind to the target in theabove BIACORE cross-blocking assay such that during the assay and in thepresence of a second amino acid sequence or other binding agent of theinvention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%)of the maximum theoretical binding, specifically between 75% and 0.1%(e.g. 75% to 4%) of the maximum theoretical binding, and morespecifically between 70% and 0.1% (e.g. 70% to 4%) of maximumtheoretical binding (as just defined above) of the two amino acidsequences or binding agents in combination. The BIACORE assay describedabove is a primary assay used to determine if amino acid sequences orother binding agents cross-block each other according to the invention.On rare occasions particular amino acid sequences or other bindingagents may not bind to target coupled via amine chemistry to a CM5BIACORE chip (this usually occurs when the relevant binding site ontarget is masked or destroyed by the coupling to the chip). In suchcases cross-blocking can be determined using a tagged version of thetarget, for example a N-terminal His-tagged version. In this particularformat, an anti-His amino acid sequence would be coupled to the BIACOREchip and then the His-tagged target would be passed over the surface ofthe chip and captured by the anti-His amino acid sequence. The crossblocking analysis would be carried out essentially as described above,except that after each chip regeneration cycle, new His-tagged targetwould be loaded back onto the anti-His amino acid sequence coatedsurface. In addition to the example given using N-terminal His-taggedtarget, C-terminal His-tagged target could alternatively be used.Furthermore, various other tags and tag binding protein combinationsthat are known in the art could be used for such a cross-blockinganalysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAGantibodies; biotin tag with streptavidin).

The following generally describes an ELISA assay for determining whetheran amino acid sequence or other binding agent directed against a targetcross-blocks or is capable of cross-blocking as defined herein. It willbe appreciated that the assay can be used with any of the amino acidsequences (or other binding agents such as polypeptides of theinvention) described herein. The general principal of the assay is tohave an amino acid sequence or binding agent that is directed againstthe target coated onto the wells of an ELISA plate. An excess amount ofa second, potentially cross-blocking, anti-target amino acid sequence isadded in solution (i.e. not bound to the ELISA plate). A limited amountof the target is then added to the wells. The coated amino acid sequenceand the amino acid sequence in solution compete for binding of thelimited number of target molecules. The plate is washed to remove excesstarget that has not been bound by the coated amino acid sequence and toalso remove the second, solution phase amino acid sequence as well asany complexes formed between the second, solution phase amino acidsequence and target. The amount of bound target is then measured using areagent that is appropriate to detect the target. An amino acid sequencein solution that is able to cross-block the coated amino acid sequencewill be able to cause a decrease in the number of target molecules thatthe coated amino acid sequence can bind relative to the number of targetmolecules that the coated amino acid sequence can bind in the absence ofthe second, solution phase, amino acid sequence. In the instance wherethe first amino acid sequence, e.g. an Ab-X, is chosen to be theimmobilized amino acid sequence, it is coated onto the wells of theELISA plate, after which the plates are blocked with a suitable blockingsolution to minimize non-specific binding of reagents that aresubsequently added. An excess amount of the second amino acid sequence,i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Ytarget binding sites per well are at least 10 fold higher than the molesof Ab-X target binding sites that were used, per well, during thecoating of the ELISA plate. Target is then added such that the moles oftarget added per well are at least 25-fold lower than the moles of Ab-Xtarget binding sites that were used for coating each well. Following asuitable incubation period the ELISA plate is washed and a reagent fordetecting the target is added to measure the amount of targetspecifically bound by the coated anti-target amino acid sequence (inthis case Ab-X). The background signal for the assay is defined as thesignal obtained in wells with the coated amino acid sequence (in thiscase Ab-X), second solution phase amino acid sequence (in this caseAb-Y), target buffer only (i.e. without target) and target detectionreagents. The positive control signal for the assay is defined as thesignal obtained in wells with the coated amino acid sequence (in thiscase Ab-X), second solution phase amino acid sequence buffer only (i.e.without second solution phase amino acid sequence), target and targetdetection reagents. The ELISA assay may be run in such a manner so as tohave the positive control signal be at least 6 times the backgroundsignal. To avoid any artefacts (e.g. significantly different affinitiesbetween Ab-X and Ab-Y for the target) resulting from the choice of whichamino acid sequence to use as the coating amino acid sequence and whichto use as the second (competitor) amino acid sequence, thecross-blocking assay may be run in two formats: 1) format 1 is whereAb-X is the amino acid sequence that is coated onto the ELISA plate andAb-Y is the competitor amino acid sequence that is in solution and 2)format 2 is where Ab-Y is the amino acid sequence that is coated ontothe ELISA plate and Ab-X is the competitor amino acid sequence that isin solution. Ab-X and Ab-Y are defined as cross-blocking if, either informat 1 or in format 2, the solution phase anti-target amino acidsequence is able to cause a reduction of between 60% and 100%,specifically between 70% and 100%, and more specifically between 80% and100%, of the target detection signal {i.e. the amount of target bound bythe coated amino acid sequence) as compared to the target detectionsignal obtained in the absence of the solution phase anti-target aminoacid sequence (i.e. the positive control wells).

The amino acid residues of a Nanobody® are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, J. Immunol.Methods 2000 Jun. 23; 240 (1-2): 185-195 (see for example FIG. 2 of thispublication); or referred to herein. According to this numbering, FR1 ofa Nanobody® comprises the amino acid residues at positions 1-30, CDR1 ofa Nanobody® comprises the amino acid residues at positions 31-35, FR2 ofa Nanobody® comprises the amino acids at positions 36-49, CDR2 of aNanobody® comprises the amino acid residues at positions 50-65, FR3 of aNanobody® comprises the amino acid residues at positions 66-94, CDR3 ofa Nanobody® comprises the amino acid residues at positions 95-102, andFR4 of a Nanobody® comprises the amino acid residues at positions103-113. [In this respect, it should be noted that—as is well known inthe art for V_(H) domains and for V_(HH) domains—the total number ofamino acid residues in each of the CDR's may vary and may not correspondto the total number of amino acid residues indicated by the Kabatnumbering (that is, one or more positions according to the Kabatnumbering may not be occupied in the actual sequence, or the actualsequence may contain more amino acid residues than the number allowedfor by the Kabat numbering). This means that, generally, the numberingaccording to Kabat may or may not correspond to the actual numbering ofthe amino acid residues in the actual sequence. Generally, however, itcan be said that, according to the numbering of Kabat and irrespectiveof the number of amino acid residues in the CDR's, position 1 accordingto the Kabat numbering corresponds to the start of FR1 and vice versa,position 36 according to the Kabat numbering corresponds to the start ofFR2 and vice versa, position 66 according to the Kabat numberingcorresponds to the start of FR3 and vice versa, and position 103according to the Kabat numbering corresponds to the start of FR4 andvice versa.]. Alternative methods for numbering the amino acid residuesof V_(H) domains, which methods can also be applied in an analogousmanner to V_(HH) domains from Camelids and to Nanobodies®, are themethod described by Chothia et al. (Nature 342, 877-883 (1989)), theso-called “AbM definition” and the so-called “contact definition”.However, in the present description, claims and figures, the numberingaccording to Kabat as applied to V_(HH) domains by Riechmann andMuyldermans will be followed, unless indicated otherwise.

By the term “target molecule” or “target molecules” or “target” is meanta protein with a biological function in an organism including bacteriaand virus, preferably animal, more preferably mammal most preferredhuman, wherein said biological function may be involved in theinitiation or progression or maintenance of a disease.

The terms “stability” and “stable” as used herein in the context of aformulation comprising a polypeptide comprising one or more singlevariable domains refer to the resistance of the polypeptide in theformulation to aggregation (and particularly dimerization and/oroligomerization) under given storage conditions. Apart from this and/orin addition, the “stable” formulations of the invention retainbiological activity under given storage conditions. The stability ofsaid polypeptide can be assessed by degrees of aggregation (andparticularly dimerization and/or oligomerization; as measured e.g. bySE-HPLC), and/or by % of biological activity (as measured e.g. by ELISA,BIACORE, etc.) compared to a reference formulation. For example, areference formulation may be a reference standard frozen at −20° C. or<−65° C. (such as e.g. −80° C.) consisting of the same polypeptide atthe same concentration in D-PBS or consisting of the same polypeptide atthe same concentration and in the same buffer as the stressed samplesbut without applying the stress conditions, which reference formulationregularly gives a single peak by SE-HPLC and/or keeps its biologicalactivity in BIACORE and/or ELISA.

The term “very little to no loss of the biological activities” as usedherein refers to single variable domain activities, including but notlimited to, specific binding abilities of the single variable domain tothe target of interest as measured by various immunological assays,including, but not limited to ELISAs and/or by Surface Plasmon Resonance(BIACORE). In one embodiment, the single variable domains of theformulations of the invention retain at least 50%, preferably at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% or more ofthe ability to specifically bind to an antigen as compared to areference formulation, as measured by an immunological assay known toone of skill in the art or described herein. For example, an ELISA basedassay (e.g. as described in the Example section) may be used to comparethe ability of the single variable domain to specifically bind to itstarget. A “reference formulation” as used herein refers to a formulationthat is frozen at a temperature of −20±5° C. or at <−64° C. (such ase.g. at −80° C.) consisting of the same single variable domain at thesame concentration in D-PBS or consisting of the same single variabledomains at the same concentration in the same buffer/excipients as thestressed samples but without applying the stress conditions, whichreference formulation regularly gives a single peak by SE-HPLC and/orkeeps its biological activity in BIACORE and/or ELISA.

The phrase “pharmaceutically acceptable” as used herein means approvedby a regulatory agency of the Federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopoeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. In this sense, it should be compatible with the otheringredients of the formulation and not eliciting an unacceptabledeleterious effect in the subject.

As used herein, the term “effective amount” refers to the amount of anagent (e.g. a prophylactic or therapeutic agent) which is sufficient toreduce and/or ameliorate the severity and/or duration of one or morediseases and/or disorders.

The term “polyol” as used herein refers to sugars that contains manyhydroxyl (—OH) groups compared to a normal saccharide. Polyols includealcohols and carbohydrates such as mannitol, sorbitol, maltitol,xylitol, isomalt, erythritol, lactitol, sucrose, glucose, galactose,fructose, fucose, ribose, lactose, maltose and cellubiose.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the prevention, treatmentand/or management of one ore more diseases and/or disorders. In thecontext of the present invention, the term “therapeutic agent” refers toa polypeptide comprising one or more single variable domains. In certainother embodiments, the term “therapeutic agent” refers to an agent otherthan the polypeptide of the invention which might be used in theformulation.

As used herein, the term “therapeutically effective amount” refers tothe amount of a therapeutic agent (e.g. a polypeptide comprising one ormore single variable domains), that is sufficient to reduce the severityof one or more diseases and/or disorders.

The term “excipient” as used herein refers to an inert substance whichis commonly used as a diluent, vehicle, preservative, binder orstabilizing agent for drugs which imparts a beneficial physical propertyto a formulation, such as increased protein stability, increased proteinsolubility, and/or decreased viscosity. Examples of excipients include,but are not limited to, proteins (e.g., serum albumin), amino acids(e.g., aspartic acid, glutamic acid, lysine, arginine, glycine),surfactants (e.g., SDS, TWEEN (polysorbate) 20, TWEEN (polysorbate) 80,poloxamers, polysorbate and nonionic surfactants), saccharides (e.g.,glucose, sucrose, maltose and trehalose), polyols (e.g., mannitol andsorbitol), fatty acids and phospholipids (e.g., alkyl sulfonates andcaprylate). For additional information regarding excipients, seeRemington's Pharmaceutical Sciences (by Joseph P. Remington, 18th ed.,Mack Publishing Co., Easton, Pa.), which is incorporated herein in itsentirety.

The term “variable domain” refers to the part or domain of animmunoglobulin molecule or antibody which is partially or fullyresponsible for antigen binding. The term “single variable domain” or“immunoglobulin single variable domain” (used interchangeably), definesmolecules wherein the antigen binding site is present on, and formed by,a single immunoglobulin domain. This sets single variable domains apartfrom “conventional” immunoglobulins or their fragments, wherein twoimmunoglobulin domains, in particular two “variable domains” interact toform an antigen binding site. Typically, in conventionalimmunoglobulins, a heavy chain variable domain (VH) and a light chainvariable domain (VL) interact to form an antigen binding site. In thiscase, the complementarity determining regions (CDRs) of both VH and VLwill contribute to the antigen binding site, i.e. a total of 6 CDRs willbe involved in antigen binding site formation.

In contrast, the binding site of a single variable domain is formed by asingle VH or VL domain. Hence, the antigen binding site of a singlevariable domain is formed by no more than three CDRs. The term “singlevariable domain” does comprise fragments of conventional immunoglobulinswherein the antigen binding site is formed by a single variable domain.

The single variable domains that are present in the constructs of theinvention may be any variable domain that forms a single antigen bindingunit. Generally, such single variable domains will be amino acidsequences that essentially consist of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively); or any suitable fragment of such an amino acid sequence(which will then usually contain at least some of the amino acidresidues that form at least one of the CDR's, as further describedherein). Such single variable domains and fragments are most preferablysuch that they comprise an immunoglobulin fold or are capable forforming, under suitable conditions, an immunoglobulin fold. As such, thesingle variable domain may for example comprise a light chain variabledomain sequence (e.g. a V_(L)-sequence) or a suitable fragment thereof;or a heavy chain variable domain sequence (e.g. a V_(H)-sequence orV_(HH) sequence) or a suitable fragment thereof; as long as it iscapable of forming a single antigen binding unit (i.e. a functionalantigen binding unit that essentially consists of the single variabledomain, such that the single antigen binding domain does not need tointeract with another variable domain to form a functional antigenbinding unit, as is for example the case for the variable domains thatare present in for example conventional antibodies and ScFv fragmentsthat need to interact with another variable domain—e.g. through aV_(H)/V_(L) interaction—to form a functional antigen binding domain).

In one aspect of the invention, the single variable domains are lightchain variable domain sequences (e.g. a V_(L)-sequence), or heavy chainvariable domain sequences (e.g. a V_(H)-sequence); more specifically,the single variable domains can be heavy chain variable domain sequencesthat are derived from a conventional four-chain antibody or heavy chainvariable domain sequences that are derived from a heavy chain antibody.

For example, the single variable domain may be a domain antibody (or anamino acid sequence that is suitable for use as a domain antibody), asingle domain antibody (or an amino acid sequence that is suitable foruse as a single domain antibody), a “dAb” or dAb (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody® (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 04/068820, WO06/030220, WO 06/003388 and other published patent applications ofDomantis Ltd. It should also be noted that, although less preferred inthe context of the present invention because they are not of mammalianorigin, single domain antibodies or single variable domains can bederived from certain species of shark (for example, the so-called “IgNARdomains”, see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody® or a suitable fragment thereof. [Note: Nanobody®, Nanobodies®and Nanoclone® are trademarks of Ablynx N.V.] For a further descriptionof V_(HH)'s and Nanobodies®, reference is made to the review article byMuyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; aswell as to the following patent applications, which are mentioned asgeneral background art: WO 94/04678, WO 95/04079 and WO 96/34103 of theVrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie(VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 bythe National Research Council of Canada; WO 03/025020 (=EP 1 433 793) bythe Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V.and the further published patent applications by Ablynx N.V. Referenceis also made to the further prior art mentioned in these applications,and in particular to the list of references mentioned on pages 41-43 ofthe International application WO 06/040153, which list and referencesare incorporated herein by reference. As described in these references,Nanobodies® (in particular V_(HH) sequences and partially humanizedNanobodies®) can in particular be characterized by the presence of oneor more “Hallmark residues” in one or more of the framework sequences. Afurther description of the Nanobodies®, including humanization and/orcamelization of Nanobodies®, as well as other modifications, parts orfragments, derivatives or “Nanobody® fusions”, multivalent constructs(including some non-limiting examples of linker sequences) and differentmodifications to increase the half-life of the Nanobodies® and theirpreparations can be found e.g. in WO07/104529, WO 08/101985 and WO08/142164.

The total number of amino acid residues in a Nanobody can be in theregion of 110-120, is preferably 112-115, and is most preferably 113. Itshould however be noted that parts, fragments, analogs or derivatives(as further described herein) of a Nanobody are not particularly limitedas to their length and/or size, as long as such parts, fragments,analogs or derivatives meet the further requirements outlined herein andare also preferably suitable for the purposes described herein.

Thus, in the meaning of the present invention, the term “single variabledomain” comprises polypeptides which are derived from a non-humansource, preferably a camelid, preferably a camelid heavy chain antibody.They may be humanized, as previously described. Moreover, the termcomprises polypeptides derived from non-camelid sources, e.g. mouse orhuman, which have been “camelized”, as previously described.

In a specific aspect, the “single variable domain” is a “single variableVHH domain”. The term “single variable VHH domain” indicates that the“single variable domain” is derived from a heavy chain antibody,preferably a camelid heavy chain antibody.

The term “single variable domain” also encompasses variable domains ofdifferent origin, comprising mouse, rat, rabbit, donkey, human andcamelid variable domains; as well as fully human, humanized or chimericvariable domains. For example, the invention comprises camelid variabledomains and humanized camelid variable domains, or camelized variabledomains, e.g. camelized dAb as described by Ward et al (see for exampleWO 94/04678 and Davies and Riechmann (1994, FEBS Lett. 339(3): 285-290)and (1996, Protein Eng. 9(6): 531-537)). Moreover, the inventioncomprises fused variable domains, e.g. multivalent and/or multispecificconstructs (for multivalent and multispecific polypeptides containingone or more V_(HH) domains and their preparation, reference is also madeto Conrath et al. 2001 (J. Biol. Chem. 276: 7346-7350) as well as to forexample WO 96/34103 and WO 99/23221).

Unless indicated otherwise, the term “immunoglobulin sequence”—whetherused herein to refer to a heavy chain antibody or to a conventional4-chain antibody—is used as a general term to include both the full-sizeantibody, the individual chains thereof, as well as all parts, domainsor fragments thereof (including but not limited to antigen-bindingdomains or fragments such as V_(HH) domains or V_(H)/V_(L) domains,respectively). The terms antigen-binding molecules or antigen-bindingprotein are used interchangeably with immunoglobulin sequence, andinclude Nanobodies.

The single variable domains provided by the invention are preferably inessentially isolated form (as defined herein), or form part of apolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more single variable domains and which mayoptionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more single variable domains may beused as a binding unit in such a polypeptide, which may optionallycontain one or more further amino acid sequences that can serve as abinding unit (i.e. against one or more other targets), so as to providea monovalent, multivalent or multispecific polypeptide of the invention,respectively as e.g. described in WO 08/101985, WO 08/142164, WO09/068625, WO 09/068627 and WO 08/020079. Such a protein or polypeptidemay also be in essentially isolated form (as defined herein) and themethods of the present invention for the expression and/or production ofsingle variable domains equally apply to polypeptides comprising one ormore single variable domains.

According to the invention, the term “single variable domain” maycomprise constructs comprising two or more antigen binding units in theform of single variable domain, as outlined above. For example, two (ormore) variable domains with the same or different antigen specificitycan be linked to form e.g. a bivalent, trivalent or multivalentconstruct. By combining variable domains of two or more specificities,bispecific, trispecific etc. constructs can be formed. For example, avariable domain according to the invention may comprise two variabledomains directed against target A, and one variable domain againsttarget B. Such constructs and modifications thereof, which the skilledperson can readily envisage, are all encompassed by the term variabledomain as used herein and are also referred to as “polypeptide of theinvention” or “polypeptides of the invention”.

The polypeptide comprising one or more single variable domains for usein the formulation of the invention may be therapeutic or prophylactic,and may be useful in the treatment and/or management of one or morediseases. In one specific aspect, the polypeptide has at least onesingle variable domain. In another specific aspect, the polypeptide hasat least two single variable domains. In yet another specific aspect,the polypeptide has at least three single variable domains. Preferably,the polypeptide comprises at least one single variable domain directedagainst HSA. In another specific aspect, the polypeptide comprises atleast a single variable domain against RANKL. In another specificaspect, the polypeptide comprises at least a single variable domainagainst IL-6R. More preferably, the polypeptide is directed againstand/or specifically binds HSA as well as another target such as RANKL orIL-6R. In yet another aspect, polypeptide comprises at least a singlevariable domain against RANKL and at least a single variable domainagainst HSA. In yet another aspect, polypeptide comprises at least asingle variable domain against IL-6R and at least a single variabledomain against HSA. In yet another aspect, polypeptide comprises atleast two single variable domains against one target and at least asingle variable domain against HSA. In yet another aspect, polypeptidecomprises at least two single variable domains against RANKL and atleast a single variable domain against HSA. In yet another aspect,polypeptide comprises at least two single variable domains against IL-6Rand at least a single variable domain against HSA. In a preferredaspect, the single variable domains used in the polypeptide of theinvention are selected from WO 08/142164 (such as e.g. SEQ ID NO's: 745and/or 791 of WO 08/142164), WO 08/020079, WO 09/068627 (such as e.g.SEQ ID NO's 2578, 2584 and/or 2585 of WO 09/068627), U.S. provisionalapplication No. 61/168,379 by Ablynx N.V., U.S. provisional applicationNo. 61/168,410 by Ablynx N.V. (such as e.g. SEQ ID NO's: 77 and/or 109of U.S. 61/168,410) and WO 08/028977 (such as e.g. SEQ ID NO: 62 of WO08/028977). Preferred polypeptides of the invention are selected fromSEQ ID NO's: 7 to 12 and 17 to 20.

The term “non-fused” in the context of ‘non-fused dimers’ means everystable linkage (or also more specific conditions herein mentioned as“stable”) existing under normal (e.g. storage and/or physiological)conditions which is not obtained via a direct genetic linkage or via adedicated dimerization sequence as known in the literature (e.g. Jun-Fosinteraction, interaction of CH2-CH3 domains of heavy-chains etc). Suchlinkage may be due to for example through chemical forces such as Vander Waal's forces, hydrogen bonds, and/or forces between peptidesbearing opposite charges of amino acid residues. Furthermore, additionalcomponents such as structural changes may play a role. Such structuralchanges may e.g. be an exchange of framework regions, e.g. exchange offramework region 4 (a phenomenon also called “domain swapping pattern”)beta strands derived from framework regions and may be prevented bystabilizing CDR3-FR4 region in the monomeric structure conformation. Incontrast in a genetically linked or—fused construct, the fusion isforcing two entities to be expressed as a fusion protein, and thelinkage is of a covalent nature (e.g. using peptide linkers between thetwo entities, linking the C-terminus of one with the N-terminus of theother protein domain). The term “stable” in the context of “stabledimer” or “stable NFD” (“stable NFDs”) means that 50%, more preferably60%, more preferably 70%, more preferably 80%, even more preferably 90%,even more preferably 95%, most preferred 99% are in the form of NFDs atthe time point of measurement; wherein 100% represents the amount (e.g.molar amount per volume or weight per volume amount) of NFD and itscorresponding monomer. Measurement of stability as defined herein, i.e.with regards to its dimeric nature, may be done by using size exclusionchromatography (using standard laboratory conditions such as PBS bufferat room temperature) and if required a pre-concentration step of thesample to be tested. The area under the peak in the size exclusionchromatogram of the identified dimeric and monomeric peak represents therelative amounts of the monomer and dimer, i.e. the NFD. NFD and/or NFDsare used herein interchangeably, thus wherever NFD is used NFDs aremeant as well and vice versa.

A polypeptide or single variable domain that is “susceptible todimerization”, as used in the present invention, means that therespective polypeptide or single variable domain, under the specifiedconditions described in the present application (e.g. in a processcalled process-induced association and/or e.g. under stressful storageconditions, such as relative high temperature (e.g. 37° C.) over weeks(such as e.g. 4 weeks)), converts its otherwise stable monomeric singlevariable domains into stable dimeric molecules (i.e. NFDs as describedherein).

Non-Fused-Dimers (NFDs)

Certain conditions or amino acid sequence alterations can convertotherwise stable monomeric single variable domains into stable dimericand in certain instances multimeric molecules. Key in this process is toprovide conditions in which two single variable domains are able todisplay an increased non-covalent interaction. NFDs are made e.g. in aprocess called process-induced association (hereinafter also “PIA”).This dimerization is among others a concentration driven event and cane.g. be enhanced by combining high protein concentrations (e.g. higherthan 50 mg protein/ml), rapid pH shifts (e.g. pH shift of 2 units within1 column volume) and/or rapid salt exchanges (e.g. salt exchange with 1column volume) in the preparation process. The high concentration willenhance the likelihood of interactions of individual monomeric moleculeswhile the pH and salt changes can induce transiently (partial) unfoldingand/or promote hydrophobic interactions and/or rearrangement of theprotein structure. Because these NFDs may ultimately be used in or as atherapeutic or prognostic agent, the term “NFD” or “NFDs” are meant tomean (or to be interchanged) that the NFD is in solution, e.g. in aphysiological preparation, e.g. physiological buffer, comprising NFD orNFDs (unless the condition, e.g. a condition of special sorts, e.g.storage condition for up to 2.5 years for which a NFD is stable, isspecifically described). Alternatively, NFDs can also be made understressful storage conditions e.g. such as relative high temperature(e.g. 37° C.) over weeks such as e.g. 4 weeks. Furthermore, NFDs can bemade (even with improved, i.e. faster, kinetics) by introducingdestabilizing amino acid residues in the vicinity of the CDR3 and/or theframework region 4 of the single variable domain susceptible to dimerize(see experimental part, polypeptide F (=mutated polypeptide B) isforming NFDs more quickly than polypeptide B under the same conditions).

Attaining a high concentration of the components that have to dimerizecan be obtained with a variety of procedures that include conditionsthat partially unfold the immunoglobulinic structure of the singlevariable domains, e.g. Nanobodies®, e.g. via chromatography (e.g.affinity chromatography such as Protein A, ion exchange, immobilizedmetal affinity chromatography or IMAC and Hydrophobic InteractionChromatography or HIC), temperature exposure close to the Tm of thesingle variable domain, and solvents that are unfolding peptides such as1 to 2 M Guanidinium Hydrochloride. E.g. for chromatography—during theprocess of elution of the proteins off the column using e.g. a pH shiftor salt gradient (as explained later), the NFDs can be formed. Usuallythe required concentration and/or exact method to form NFDs has to bedetermined for each polypeptide of the invention and may not be possiblefor each polypeptide of the invention. It is our experience that thereare certain single variable domains either alone (e.g. polypeptides Band F) and/or in a construct (e.g. polypeptides A, C, E, F) that form aNFD. Critical for dimerization may be a relative short CDR3 (e.g. 3 to 8amino acids, more preferably 4 to 7 amino acids, even more preferably 5to 6 amino acids, e.g. 6 amino acids) and destabilizing factors in thevicinity of the CDR3 and/or FR4. Furthermore, high concentration such ase.g. the maximum solubility of the polypeptides comprising singlevariable domain(s) at the concentration used (e.g. 5 mg polypeptide Aper ml protein A resin—see experimental part), or storage at hightemperature over weeks (e.g. 37° C. over 4 weeks), low pH (e.g. pH belowpH 6), high concentration (higher than 50 mg/ml, e.g. 65 mg/ml) may berequired to obtain a reasonable yield of NFD formation.

Next to column chromatography working at e.g. maximum column load,similar required high concentration to obtain NFDs can be achieved byconcentration methods such as ultrafiltration and/or diafiltration, e.g.ultrafiltration in low ionic strength buffer.

The process is not linked to a specific number of single variabledomains, as the formation of NFDs was observed with monovalent, bivalentand trivalent monomeric building blocks (=polypeptides comprising singlevariable domain(s)) and even with single variable domain-HSA fusions. Incase the polypeptides comprises 2 different single variable domains,NFDs may form via only the identical or different (preferably theidentical) single variable domain and usually only via one of the singlevariable domain(s), e.g. the one identified as susceptible to form NFDs(e.g. polypeptide B)(see also FIG. 53b ).

It is an object of the present invention to provide soluble and stable(e.g. stable within a certain concentration range, buffer and/ortemperature conditions) dimer-complexes called NFDs that may be used totarget molecules and/or thus inhibit or promote cell responses. Hereindescribed are NFDs comprising monomeric building blocks such as singlevariable domain—also called NFDs-Mo; NFDs comprising dimeric buildingblocks such as two covalently linked single variable domains—also calledNFDs-Di; NFDs comprising trimeric building blocks such as threecovalently linked single variable domains—also called NFDs-Tri; NFDscomprising tetrameric building blocks such as four covalently linkedsingle variable domains—also called NFDs-Te; and NFDs comprising morethan four (=multimeric) building blocks such as multimeric covalentlylinked single variable domains—also called NFDs-Mu (see FIG. 53 a+b forschematic overview of such structures). The NFDs may contain identicalsingle variable domains or different single variable domains (FIG. 53b). If the building blocks (polypeptide) consist of different singlevariable domains, e.g. Nanobodies®, it is our experience that preferablyonly one of the single variable domain in the polypeptide will dimerize.E.g. the dimerizing unit (single variable domain, e.g. Nanobody® such ase.g. polypeptide B or F) of a trivalent polypeptide (see FIG. 53b ) maybe in the middle, at the C-terminus or at the N-terminus of theconstruct.

It is another object of the invention to provide methods of making anduses of said NFDs.

It is still another object of the present invention to provideinformation on how to avoid such NFDs.

These above and other objectives are provided for by the presentinvention which, in a broad sense, is directed to methods, kits,non-fused-dimers that may be used in the treatment of neoplastic, immuneor other disorders. To that end, the present invention provides forstable NFDs comprising a single variable domain or single variabledomains such as e.g. Nanobody® or Nanobodies® (e.g. polypeptide B) thatmay be used to treat patients suffering from a variety of disorders. Inthis respect, the NFDs of the present invention have been surprisinglyfound to exhibit biochemical characteristics that make them particularlyuseful for the treatment of patients, for the diagnostic assessment of adisease in patients and/or disease monitoring assessment in patients inneed thereof. More specifically, it was unexpectedly found that certainsingle variable domains, subgroups thereof (including humanized VHHs ortruly camelized human VHs) and formatted versions thereof (and indeedthis is also feasible for human VH and derivatives thereof), can be madeto form stable dimers (i.e. NFD-Mo, NFD-Di, NFD-Tri, NFD-Te or NFD-Mu)that have beneficial properties with regard e.g. to manufacturabilityand efficacy. Single variable domains are known to not denature upon forexample temperature shift but they reversibly refold upon coolingwithout aggregation (Ewert et al Biochemistry 2002, 41:3628-36), ahallmark which could contribute to efficient formation ofantigen-binding dimers.

NFDs are of particular advantage in many applications. In therapeuticapplications, NFDs-Mu, e.g. NDF-Di, binders may be advantageous insituation where oligomerization of the targeted receptors is needed suchas e.g. for the death receptors (also referred to as TRAIL receptor).E.g. a NFD-Di due to their close interaction of the respective buildingblocks are assumed to have a different spatial alignment than“conventional” covalently linked corresponding tetramers and thus mayprovide positive or negative effect on the antigen-binding (see FIG. 53for a schematic illustration of certain NFDs). Furthermore, a NFDs, e.g.a NFD-Mo, may bind a multimeric target molecule more effectively than aconventional covalently linked single variable domain dimer. Moreover,heteromeric NFDs may comprise target specific binders and binders toserum proteins, e.g. human serum albumin, with long half life. Inaddition, “conventional” covalently linked dimers (via e.g. amino acidsequence linkers) may have expression problems (by not having enoughtRNA available for certain repetitive codons) and thus it may beadvantageous to make the monomers first and than convert the monomers toa NFD in a post-expression process, e.g. by a process described herein.This may give yields that are higher for the NFD compared to thecovalently linked dimer. Similarly, it may be expected that e.g. theoverall yield of a NFD-Di or NFD-Tri will be higher compared to therelevant covalently linked tetramer or hexamer. The overall higherexpression level may be the overriding factor in e.g. cost determinationto select the NFD approach. E.g. it is reported that expression yieldsand secretion efficiency of recombinant proteins are a function of chainsize (Skerra & Pluckthun, 1991, Protein Eng. 4, 971). Moreover, lesslinker regions could mean less protease susceptible linker regions onthe overall protein. It could also be useful to test in vitro and/or invivo the impact of multimerization of a single variable domain accordingto the methods described herein. All in all, it is expected that thefinding of this invention may provide additional effective solutions inthe drug development using formatted single variable domains as theunderlying scaffold structure than with the hitherto known approaches,i.e. mainly covalently linked single variable domain formats.

The NFDs of the present invention can be stable in a desirable range ofbiological relevant conditions such as a wide range of concentration(i.e. usually low nM range), temperature (37 degrees Celsius), time(weeks, e.g. 3 to 4 weeks) and pH (neutral, pH5, pH6 or in stomach pHsuch as pH 1). In a further embodiment, NFDs of the present inventioncan be stable (at a rate of e.g. 95% wherein 100% is the amount ofmonomeric and dimeric form) in vivo, e.g. in a human body, over aprolonged period of time, e.g. 1 to 4 weeks or 1 to 3 months, and up to6 to 12 months. Furthermore, the NFDs of the present invention can alsobe stable in a desirable range of storage relevant conditions such as awide range of concentration (high concentration such as e.g. mg per mlrange), temperature (−20 degrees Celsius, 4 degrees Celsius, 20 or 25degrees Celsius), time (months, years), resistance to organic solventsand detergents (in formulations, processes of obtaining formulations).Furthermore, it has been surprisingly found that denaturation withguanidine HCl (GdnHCl) needs about 1 M more GdnHCl to denature thepolypeptide B dimer than the polypeptide B monomer in otherwise sameconditions (see experimental part). Additionally, the surprising findingthat FR4 in the polypeptide B NFD-Mo is swapped (and possibly similarlyfor other NFDs according to the invention) indicates that indeed thisdimers form stable complexes and can further stabilize single variabledomain or Nanobody® structures. Furthermore, there is evidence that oneof the humanisation sites (see experimental part: polypeptide E vs.polypeptide B) may have caused a weaker CDR3 interaction with theframework and thus a more extendable CDR3 is available that is morelikely to trigger dimerization.

Thus, preferred NFDs of the invention are stable (with regards to thedimeric nature) within the following ranges (and wherein said ranges mayfurther be combined, e.g. 2, 3, 4 or more ranges combined as describedbelow, to form other useful embodiments):

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under physiological temperature conditions, i.e. temperaturearound 37 degrees Celsius, over a prolonged time period, e.g. a time upto 1 day, more preferably 1 week, more preferably 2 weeks, even morepreferably 3 weeks, most preferred 4 weeks from the time point ofdelivery of the drug to the patient in need;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under various storage temperature conditions, i.e. temperaturessuch as −20 degrees Celsius, more preferably 4 degrees Celsius, morepreferably 20 degrees Celsius, most preferably 25 degrees Celsius, overa prolonged time period, e.g. up to 6 months, more preferably 1 year,most preferred 2 years;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under various physiological pH conditions, i.e. pH ranges suchas pH 6 to 8, more preferably pH 5 to 8, most preferred pH 1 to 8, overa prolonged time period, e.g. a time up to 1 week, more preferably 2weeks, even more preferably 3 weeks, most preferred 4 weeks from thetime point of delivery of the drug to the patient in need;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under various physiological concentration conditions, i.e.concentration of NFDs below 200 ng NFD/ml solvents, e.g. in pH 7 buffersuch as phosphate buffered solution and/or e.g. also serum, e.g. humanserum; more preferably below 100 ng NFD/ml solvents, even preferablybelow 50 ng NFD/ml solvents, most preferred 10 ng NFD/ml solvents; in afurther preferred embodiment NFDs are stable in above concentrations at37 degrees Celsius up to 1 day and more, e.g. 1 week, more preferably 2weeks, more preferably 3 weeks, and most preferred up to 4 weeks;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under various physiological concentration conditions, i.e.concentration of NFDs of about 1 mg/ml, more preferably 5 mg/ml, morepreferably 10 mg/ml, more preferably 15 mg/ml, more preferably 20 mg/ml,more preferably 30 mg/ml, more preferably 40 mg/ml, more preferably 50mg/ml, more preferably 60 mg/ml, more preferably 70 mg/ml, and attemperature around 37 degrees Celsius, over a prolonged time period,e.g. a time up to 1 day, more preferably 1 week, more preferably 2weeks, even more preferably 3 weeks, most preferred 4 weeks from thetime point of delivery of the drug to the patient in need;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) under various storage concentration conditions, i.e.concentration of NFDs above 0.1 mg NFD/ml solvents, e.g. in pH 7 buffersuch as phosphate buffered solution; more preferably above 1 mg NFD/mlsolvents; more preferably above 5 mg NFD/ml solvents; more preferablyabove 10 mg NFD/ml solvents, and most preferred above 20 mg NFD/mlsolvents; in a further preferred embodiment NFDs are stable in aboveconcentrations at −20 degree Celsius up to 6 months and more, e.g. 1year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years; in a further preferred embodiment NFDs arestable in above concentrations at 4 degrees Celsius up to 6 months andmore, e.g. 1 year, more preferably 2 years, more preferably 3 years, andmost preferred up to 4 years; in a further preferred embodiment NFDs arestable in above concentrations at 25 degrees Celsius up to 6 months andmore, e.g. 1 year, more preferably 2 years, more preferably 3 years, andmost preferred up to 4 years;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) in mixtures (e.g. pharmaceutical formulations or processintermediates) with organic solvents, e.g. alcohols such as ethanol,isopropyl alcohol, hexanol and/or others wherein alcohol (preferablyethanol) can be added up to 5%, more preferably 10%, even morepreferably 15%, even more preferably 20%, most preferably 30%, forprolonged period of time at a particular temperature, e.g. over longstorages, such as at −20 degrees Celsius up to 6 months and more, e.g. 1year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years; in a further preferred embodiment NFDs arestable in above mixtures at 4 degrees Celsius up to 6 months and more,e.g. 1 year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years; in a further preferred embodiment NFDs arestable in above mixtures at 25 degrees Celsius up to 6 months and more,e.g. 1 year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years, wherein organic solvents such as e.g. alcohol(preferably ethanol) can be added up to 5%, more preferably 10%, evenmore preferably 15%, even more preferably 20%, most preferably 30%;

Preferred embodiments of NFDs are stable (with regards to the dimericnature) in mixtures (e.g. pharmaceutical formulations or processintermediates) with detergents, e.g. non-ionic detergents such as e.g.TRITON-X, up to 0.01%, more preferably 0.1%, most preferably 1%, forprolonged period of time at a particular temperature, e.g. over longstorages, such as at −20 degrees Celsius up to 6 months and more, e.g. 1year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years; in a further preferred embodiment NFDs arestable in above mixtures at 4 degrees Celsius up to 6 months and more,e.g. 1 year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years; in a further preferred embodiment NFDs arestable in above mixtures at 25 degrees Celsius up to 6 months and more,e.g. 1 year, more preferably 2 years, more preferably 3 years, and mostpreferred up to 4 years.

Another embodiment of the current invention is that the NFDs retain thebinding affinity of at least one of the two components compared to themonomers, e.g. said affinity of the NFDs may be not less than 10%, morepreferably not less than 50%, more preferably not less than 60%, morepreferably not less than 70%, more preferably not less than 80%, or evenmore preferably not less than 90% of the binding affinity of theoriginal monomeric polypeptide; or it has multiple functional bindingcomponents, with apparent affinity improved compared to the monomer,e.g. it may have a 2 fold, 3, 4, 5, 6, 7, 8, 9 or 10 fold, morepreferably 50 fold, more preferably 100 fold more preferably 1000 foldimproved affinity compared to the original monomeric polypeptide.

Another embodiment of the current invention is that the NFDs partiallyor fully lose the binding affinity of at least one of the two componentscompared to the monomers, e.g. said affinity of the NFDs may be not lessthan 90%, more preferably not less than 80%, more preferably not lessthan 70%, more preferably not less than 60%, more preferably not lessthan 50%, even more preferably not less than 30%, even more preferablynot less than 20%, even more preferably not less than 10%, or even morepreferably not less than 1% of the binding affinity of the originalmonomeric polypeptide or most preferred the binding affinity may not bedetectable at all; or it has multiple functional binding components,with apparent affinity compared to the monomer that is decreased, e.g.it may have a 2 fold, 3, 4, 5, 6, 7, 8, 9 or 10 fold, more preferably 50fold, more preferably 100 fold more preferably 1000 fold decreasedaffinity compared to the original monomeric polypeptide.

Furthermore, an embodiment of the current invention is a preparationcomprising NFDs and their monomeric building blocks, e.g. preparationscomprising more than 30% NFDs (e.g. the 2 identical monomeric buildingblocks that form said NFD), e.g. more preferably preparations comprisingmore than 35% NFDs, even more preferably preparations comprising morethan 40% NFDs, even more preferably preparations comprising more than50% NFDs, even more preferably preparations comprising more than 60%NFDs, even more preferably preparations comprising more than 70% NFDs,even more preferably preparations comprising more than 80% NFDs, evenmore preferably preparations comprising more than 90% NFDs, even morepreferably preparations comprising more than 95% NFDs, and/or mostpreferred preparations comprising more than 99% NFDs (wherein 100%represents the total amount of NFDs and its corresponding monomericunit). In a preferred embodiment, said ratios in a preparation can bedetermined as e.g. described herein for NFDs.

Moreover, another embodiment of the current invention is apharmaceutical composition comprising NFDs, more preferably comprisingmore than 30% NFDs (e.g. the 2 identical monomeric building blocks formsaid NFD), e.g. more preferably a pharmaceutical composition comprisingmore than 35% NFDs, even more preferably a pharmaceutical compositioncomprising more than 40% NFDs, even more preferably a pharmaceuticalcomposition comprising more than 50% NFDs, even more preferably apharmaceutical composition comprising more than 60% NFDs, even morepreferably a pharmaceutical composition comprising more than 70% NFDs,even more preferably a pharmaceutical composition comprising more than80% NFDs, even more preferably a pharmaceutical composition comprisingmore than 90% NFDs, even more preferably a pharmaceutical compositioncomprising more than 95% NFDs, and/or most preferred a pharmaceuticalcomposition comprising more than 99% NFDs (wherein 100% represents thetotal amount of NFDs and its corresponding monomeric unit).

Another embodiment of the present invention is a mixture comprisingpolypeptides in monomeric and dimeric form, i.e. the NFDs, wherein saidpreparation is stable for 1 months at 4 degrees Celsius in a neutral pHbuffer in a 1 mM, more preferably 0.1 mM, more preferably 0.01 mM, morepreferably 0.001 mM, or most preferably 100 nM overall concentration(=concentration of monomeric and dimeric form), and wherein saidpreparation comprises more than 25%, more preferably 30%, morepreferably 40%, more preferably 50%, more preferably 60%, morepreferably 70%, more preferably 80% or more preferably 90% dimer, i.e.NFD.

While the methodology described here is or may be in principleapplicable to dimerize or multimerize either Fab fragments, Fvfragments, scFv fragments or single variable domains, it is the latterfor which their use is most advantageous. In this case dimericfragments, i.e. the NFDs, can be constructed that are stable, welldefined and extend the applicability of said single variable domainsbeyond the current horizon. In a preferred embodiment, the NFDs areobtainable from naturally derived VHH, e.g. from llamas or camels,according to the methods described herein or from humanized versionsthereof, or humanized versions wherein one or more of the so calledhallmark residues, e.g. the ones forming the former light chaininterface residues, also e.g. described in WO 2006/122825, or in FIG. 52herein, are not changed and stay as derived from the naturally obtainedsingle variable domains. In a further preferred embodiment, the NFDs areobtainable from polypeptides comprising at least a single domainantibody (or Nanobody®) with similar CDR3 and FR4 amino acid residues(SEQ ID NO: 14) as polypeptide B, e.g. NFDs obtainable from polypeptidescomprising at least a Nanobody® having a CDR3 and FR4 region that has a80%, more preferably 90%, even more preferably 95%, 96%, 97%, 98%, 99%sequence identity to SEQ ID NO: 14.

Previously, increasing the number of binding sites based on singlevariable domains meant the preparation of covalently linked domains atthe genetic level or via other interaction domains (e.g. via fusion toFc, Jun-Fos, CH2/CH3 constant domain of heavy chain interaction, VL-VHantibody domain interactions etc), whereas now it is possible toalternatively form such entities later, at the protein level. Thesenon-fused dimers combine three main features: (a) possibility to combineone or more single variable domains of one or more specificities (e.g.against a target molecule and against a serum protein with long halflife) into NFDs by biochemical methods (vs. genetic methods), (b)controlled dimeric interaction that retains or abolishes antigen binding(vs. “uncontrolled” aggregation), and (c) stability sufficient e.g. forlong term storage (for practical and economic reasons) and applicationin vivo, i.e. for application over prolonged time at e.g. 37 degreesCelsius (important requirement for the commercial use of these NFDs).

Thus, it is a further object of the invention to create new individualand stable NFDs with bi- or even multifunctional binding sites. It hasbeen found that antibody fragment fusion proteins containing singlevariable domains could be produced by biochemical methods which e.g.show the specified and improved properties as described herein. Forexample, a particular embodiment of the present invention is a NFD orNFDs comprising a first polypeptide comprising single variabledomain(s), e.g. a Nanobody® or Nanobodies®, against a target moleculeand a second polypeptide comprising single variable domain(s), e.g. aNanobody® or Nanobodies®, against a serum protein, e.g. human serumalbumin (see e.g. polypeptide C and E (each binding a receptor targetand human serum albumin) in the experimental part, see also FIG. 53a+b). Other examples of using bispecificity can be found in Kufer et al,Trends in Immunology 22: 238 (2004). In the case in which two differentantigen-binding single variable domains are used, the procedure toproduce NFDs may be tweaked to promote the formation of heterodimersversus homodimers, or alternatively be followed by a procedure toseparate these forms.

Moreover, it is an object of the invention, therefore, to provide (orselect) in a first step a monomeric polypeptide essentially consistingof a single variable domain, wherein said polypeptide is capable todimerize with itself by process-induced association (PIA) or otheralternative methods described herein.

More specifically, we describe in this invention NFDs obtainable by e.g.a method that comprises the step of screening for preparationscomprising antibody fragments or polypeptides comprising single variabledomain(s) that form dimers by the processes as described herein. Hencesaid screening method comprising identifying said polypeptides may be afirst step in the generation of NFDs. Multiple ‘PIA’ methods describedherein can be used to force dimer formation in a starting preparationcomprising its monomeric building block(s). An indication that dimersmay be formed under suitable conditions, e.g. the process inducedassociation (PIA) as described herein, is sufficient at this time andmay simply mean that a small amount of e.g. the protein A purifiedfraction in the size exclusion chromatography is eluting as a presumabledimer in the standard purification protocol. Once the dimerization issuggested and later confirmed (e.g. by analytical SEC, dynamic lightscattering and/or analytical ultracentrifugation) further improvement inorder to favour dimerization (e.g. by higher column load, conditionsfavouring partial unfolding, conditions favouring hydrophobicinteractions, high temperature such as e.g. 37° C. exposure of sometime, e.g. weeks such as e.g. 4 weeks, introduction of CDR3destabilizing amino acid residues etc) or in order to minimizedimerization (opposite strategy) can be initiated (in order to e.g.increase the yield).

The invention relates, furthermore, to a process of selection of amonomeric polypeptide that comprises at least one single variabledomain, preferably at least one Nanobody®, capable of forming a NFDaccording to the invention and as defined herein, characterized in thatthe NFD is stable and preferably has a similar or better apparentaffinity to the target molecule than the monomeric polypeptide showingthat the binding site is active or at least is partially active. Saidaffinity may be not less than 10%, more preferably 50%, more preferablynot less than 60%, more preferably not less than 70%, more preferablynot less than 80%, or even more preferably not less than 90% of thebinding affinity of the original monomeric polypeptide, e.g. may have a2 fold, 3, 4, 5, 6, 7, 8, 9 or 10 fold, more preferably 50 fold, morepreferably 100 fold more preferably 1000 fold improved apparent affinitycompared to original monomeric polypeptide. Said affinity may beexpressed by features known in the art, e.g. by dissociation constants,i.e. Kd, affinity constants, i.e. Ka, koff and/or kon values—these andothers can reasonably describe the binding strength of a NFD to itstarget molecule.

Moreover, the invention relates, furthermore, to a process of selectionof a monomeric polypeptide that comprises at least one single variabledomain, preferably at least one Nanobody®, capable of forming a NFDaccording to the invention and as defined herein, characterized in thatthe NFD is stable and preferably has no apparent affinity to the targetmolecule, e.g. human serum albumin.

Said selection may comprise the step of concentrating the preparationcomprising the monomeric starting material, i.e. the polypeptidecomprising or essentially consisting of at least one single variabledomain, to high concentration, e.g. concentration above 5 mg/ml resin,by methods known by the skilled person in the art, e.g. by loading saidpolypeptide to a column, e.g. protein A column, to the near overload ofthe column capacity (e.g. up to 2 to 5 mg polypeptide per ml resinprotein A) and then optionally eluting said polypeptide with a “steep”pH shift (“steep” meaning e.g. a particular pH shift or change (e.g. adecrease or increase of 10, more preferably 100 or more preferably 1000fold of the H+ concentration) in one step (i.e. immediate buffer change)or within one, two or three (more preferably one or immediate bufferchange) column volume(s)). Furthermore, the “steep” pH shift may becombined with a selected pH change, i.e. the pH can start above or belowthe pI of the polypeptide and then change into a pH below or above thepI of said polypeptide. Alternatively, concentration of saidpolypeptides leading to NFD formation is obtainable by other means suchas e.g. immobilized metal ion affinity chromatography (IMAC), orultra-filtration. Preferably conditions are used wherein thepolypeptides of the invention are likely to unfold (extremes in pH andhigh temperature) and/or combinations of conditions favouringhydrophobic interaction such as e.g. pH changes around the pI of thepolypeptide and low salt concentration. Furthermore, the conditions usedto drive these dimers apart may be also useful to explore whendetermining further methods for producing these dimers, i.e. combiningthese procedures (e.g. 15 minutes of exposure to a temperature of about70 degrees Celsius for Polypeptide A with a high polypeptideconcentration and subsequent cooling).

Examples of methods to obtain NFDs are further described in a nonlimiting manner in the experimental part of this invention.

Another object of the invention is the process to obtain a NFDcharacterized in that the genes coding for the complete monomericpolypeptide comprising at least one single variable domain (e.g. one,two, three or four single variable domain(s)) or functional parts of thesingle variable domain(s) (e.g. as obtained by the screening methoddescribed herein) are cloned at least into one expression plasmid, ahost cell is transformed with said expression plasmid(s) and cultivatedin a nutrient solution, and said monomeric polypeptide is expressed inthe cell or into the medium, and in the case that only parts of thefusion proteins were cloned, protein engineering steps are additionallyperformed according to standard techniques.

Furthermore, another object of the invention is the process ofassociating two monomeric identical polypeptides comprising at least onesingle variable domain (e.g. one, two, three or four single variabledomain(s)) or functional parts of the single variable domain(s) to forma NFD, wherein said process comprises the step of creating anenvironment where hydrophobic interactions and/or partial refolding ofsaid polypeptides are favoured e.g. by up-concentrating a preparationcomprising the monomeric polypeptides, salting-out, adding detergents ororganic solvents, neutralizing the overall charge of said polypeptide(i.e. pH of polypeptide solution around the pI of said polypeptide orpolypeptides) and/or high temperature close to the melting temperatureof the polypeptide or the single variable domain susceptible todimerization, e.g. at temperature around 37° C. or higher e.g. 40° C.,45° C. or 50° C. or higher over a prolonged time, e.g. weeks such ase.g. 1, 2 3, 4 or more weeks, preferably 4 weeks during dimerizationprocess thus allowing close interaction between the polypeptides.Interestingly and surprisingly said conditions do not have to be upheldin order to stabilize the NFDs once the dimer is formed, i.e. the NFDsin solution are surprisingly stable in a wide range of biologicalrelevant conditions such as mentioned herein.

The NFDs according to the invention may show a high avidity againstcorresponding antigens and a satisfying stability. These novel NFDstructures can e.g. easily be prepared during the purification processfrom the mixture of polypeptides and other proteins and/or peptidesobtained by the genetically modified prokaryotic or eukaryotic host cellsuch as e.g. E. coli and Pichia pastoris.

Furthermore, the monomeric building blocks capable of forming NFDs maybe pre-selected before doing a process for selection or screening asabove and further herein described by taking into consideration primaryamino acid sequences and crystal structure information if available.Moreover, in order to understand the potential interactions in thesenon-fused protein domains, it may be advisable to analyze differentX-ray or NMR structures of non-fused single variable domains, i.e. NFDs.This then exemplifies how possibly in solution interactions in NFDs canoccur but this is by no means then a complete explanation for the likelyarea of interaction between the NFD components.

Furthermore, further stabilization of the dimer may be beneficial andmay be done by suitable linker linking the ends of the polypeptidesand/or cysteines at the interaction sites. E.g. a covalent attachment ofthe two domains may be possible by introducing 2 cysteines in each ofthe two building blocks at spatially opposite positions to forceformation of a disulphide bridge at the new site of interaction, or atN- or C-terminal region of the NFD as has e.g. been done with diabodies(Holliger & Hudson, Nat Biotech 2004, 23 (9): 1126). Furthermore, it maybe advantageous to introduce a flexible peptide between the ends of thetwo monomeric building blocks. As an example, the upper hinge region ofmouse IgG3 may be used. However, a variety of hinges or other linkersmay be used. It is not required for dimerization per se, but provides alocking of the two building blocks. The naturally occurring hinges ofantibodies are reasonable embodiments of hinges. In such case, thepolypeptides of the invention need to be present first under reducingconditions, to allow the NFDs to form during purification after whichoxidation can lead to the cysteine pairings, locking the NFDs into afixed state. In the case of NFDs, the hinges or linkers may be shorterthan in conventional covalently linked single variable domain containingpolypeptides. This is not to disturb the expected close interaction ofthe monomeric building blocks, and flexibility of the dimer is notnecessary. The choice of the hinge is governed by the desired residuesequence length (Argos, 1990, J. Mol. Biol. 211, 943-958), compatibilitywith folding and stability of the dimers (Richardson & Richardson, 1988,Science 240, 1648-1652), secretion and resistance against proteases, andcan be determined or optimized experimentally if needed.

Furthermore, further stabilization of the monomers may be beneficial(i.e. avoidance of the dimerization or in certain instances possiblemultimerizations) and may be done by choosing suitable linkers linkingthe ends of the polypeptides and/or cysteines at or close to the CDR3and/or FR4 region that prevent the single variable domain fromdimerisation. E.g. a covalent stabilization of the CDR3 and/or FR4 maybe possible by introducing 2 cysteines close to or/and within the CDR3and/or FR4 region at spatially opposite positions to force formation ofa disulphide bridge as has e.g. been done with cystatin that wasstabilized against three-dimensional domain swapping by engineereddisulfide bonds (Wahlbom et al., J. of Biological Chemistry Vol. 282,No. 25, pp. 18318-18326, Jun. 22, 2007). Furthermore, it may beadvantageous to introduce a flexible peptide that is then engineered tohave one cysteine that than forms a disulfide bond to e.g. a cysteinebefore the CDR3 region. In such case, the polypeptides of the inventionneed to be present first under reducing conditions, to allow themonomers to form after which oxidation can lead to the cysteinepairings, locking the monomers into a fixed, stabilized state.

Furthermore, further stabilization of the monomers may be beneficial(i.e. avoidance of the dimerization or in certain instances possiblemultimerizations) and may be done by replacing a destabilizing aminoacid residue or residues (e.g. identified by screening of mutants, e.g.by affinity maturation methods—see e.g. WO2009/004065) by a stabilizingamino acid residue or residues in the vicinity of CDR3 and/or FR4.

In another aspect of the invention, further stabilization of themonomers can be achieved (i.e. avoidance of the dimerization or incertain instances possible multimerizations) by suitable formulation. Inparticular, the present invention provides a method for suppressing thedimerization and multimerization of (human serum) albumin-bindingNanobodies® (e.g. polypeptide B) and other polypeptides comprisingNanobodies® by providing mannitol or other polyols to a liquidformulation. Mannitol is generally used for maintaining the stabilityand isotonicity of liquid protein formulations. It is also a commonbulking agent for lyophilization of the formulation. Surprisingly, thepresent invention discovered that mannitol can specifically inhibit theformation of dimers observed during storage (at elevated temperature) ofseveral albumin-binding Nanobodies®. As a result, mannitol-containingformulations increase protein stability and sustain biological activity,thereby prolonging the shelf-life of the drug product. The stabilizingeffect of mannitol is supported by data that demonstrate higher Tm(melting temperature) values in protein formulations with increasingmannitol concentrations.

This invention will also cover the use of other polyols, non-reducingsugars, NaCl or amino acids.

The dimers formed by e.g. the serum albumin-binding Nanobody®“polypeptide B” of the invention (SEQ ID NO: 8) was shown to becompletely inactive for binding to HSA (BIACORE analysis), suggestingthat the albumin binding site in the dimer interface is blocked by dimerformation. The addition of mannitol to the liquid formulation asproposed by this invention will therefore not only suppress thedimerization process but, importantly, will also preserve theHSA-binding activity of Nanobody® and slow down the inactivation. Ingeneral, the mannitol containing formulations according to theinventions prolong the shelf-life of the formulated protein/drugproduct. The invention is believed to be applicable to anyalbumin-binding Nanobody® and may be applicable to all Nanobodies® thathave a tendency to form dimers in general. Thus, the mannitolformulations of the invention are indicated for the formulation of anyNanobody®, as process intermediate, drug substance or drug product. Thisinvention may be used in a wide variety of liquid formulations which mayconsist of any buffering agent, a biologically effective amount ofprotein, a concentration of mannitol that is no greater thanapproximately 0.6M and other excipients including polyols, non-reducingsugars, NaCl or amino acids. The liquid formulations may be storeddirectly for later use or may be prepared in a dried form, e.g. bylyophilization. Mannitol may be used in any formulation to inhibit theformation of high molecular weight species such as the observed dimersduring storage, freezing, thawing and reconstitution afterlyophilization.

Thus, the present invention also relates to a formulation that comprisesa polypeptide comprising one or more single variable domains, saidformulation being formulated for administration to a human subject, andsaid formulation further comprising an excipient at a concentration of1% to 20% (w:v).

Preferred excipients include polyols and/or sugars. The polyol and/orsugar may be a monosaccharide such as glucose or mannose, or apolysaccharide including disaccharides such as (without being limiting)sucrose and lactose, as well as sugar derivatives including sugaralcohols and sugar acids. Polyols and sugar alcohols include (withoutbeing limiting) mannitol, xylitol, erythritol, threitol, sorbitol andglycerol. A non-limiting example of a sugar acid is L-gluconate. Otherexemplary sugars include (without being limiting) trehalose, glycine,maltose, raffinose, etc. The concentration of the excipient may rangefrom about 1% to 20% (w:v), preferably from about 2.5% to 10% (w:v),more preferably from about 5% to 10% (w:v), such as e.g. 5% (w:v), 7.5%(w:v), 8% or 10% (w:v). Throughout the present invention theconcentration of the excipient will be given as % (w:v). In a preferredaspect, the formulation comprises sucrose, preferably at a concentrationof about 5% to 10% (w:v), such as about 8% (w:v).

In one aspect, the formulation of the present invention comprises anaqueous carrier with a pH of 5.5 to 8.0 and a polypeptide comprising oneor more single variable domains at a concentration of 1 mg/ml to 200mg/ml, said formulation being formulated for administration to a humansubject, and said formulation further comprising an excipient at aconcentration of 1% to 20% (w:v).

In another aspect, the formulation of the present invention comprises anaqueous carrier with a pH of 5.5 to 8.0 and a polypeptide comprising oneor more single variable domains at a concentration of 1 mg/ml to 200mg/ml, said formulation being formulated for administration to a humansubject, and said formulation further comprising an excipient at aconcentration of 1% to 20% (w:v), wherein said formulation has aninorganic salt concentration of 150 mM or lower.

The stable formulations of the present invention comprise polypeptidesof the invention that have a high stability even during transportationand/or long periods of storage and that exhibit little to no aggregation(particularly dimerization and/or oligomerization). In addition to thepolypeptide of the invention, the formulations of the present inventioncomprise at least an aqueous carrier and a buffer. The carrier used inthe formulation of the invention should be a liquid carrier. Preferablythe carrier is an aqueous carrier such as e.g. distilled water, MILLI-Qwater or Water for Injection (WFI).

The pH of the formulation of the invention generally should not be equalto the isoelectric point of the particular polypeptide and may rangefrom about 5.5 to about 8.0, or from about 6.0 to about 7.5, preferablyfrom about 6.2 to 7.5, from about 6.5 to 7.5, most preferably from about6.5 to 7.0.

The buffer can be any pharmaceutically acceptable buffer and can(without being limiting) be e.g. selected from the group consisting ofhistidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5and acetate pH 5.5-6.0. The concentration of the buffer present in theformulation of the invention may range from 1 mM to 100 mM, 5 mM to 100mM, 5 mM to 75 mM, 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25 mM, 10 mMto 20 mM. In a specific aspect, the concentration of buffer in theformulations of the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM,25 mM, 50 mM, 75 mM, or 100 mM. Preferably, the concentration is between10 and 20 mM, such as 10 mM or 15 mM.

It will be understood by one skilled in the art that the formulation ofthe invention may be isotonic or slightly hypotonic with human blood,i.e. the formulation of the invention has essentially the same or aslightly lower osmotic pressure as human blood. Such isotonic orslightly hypotonic formulation generally has an osmotic pressure fromabout 240 mOSm/kg to about 320 mOSm/kg, such as about 240 mOSm/kg orhigher, 250 mOSm/kg or higher or 260 mOSm/kg or higher.

Tonicity of a formulation is adjusted by the use of tonicity modifiers.“Tonicity modifiers” are those pharmaceutically acceptable inertsubstances that can be added to the formulation to provide anisotonicity of the formulation. Preferred tonicity modifier in theformulation of the invention are salts and/or excipients.

The formulation of the invention may additionally comprise a surfactant.A surfactant refers to a surface-active agent comprising a hydrophobicportion and a hydrophilic portion. In a preferred aspect, the surfactantis non-ionic. Certain exemplary non-ionic surfactants include (withoutbeing limiting) PEG8000, and polysorbate, including without beinglimiting, polysorbate 80 (TWEEN 80) and polysorbate 20 (TWEEN 20),TRITON X-100, polyoxypropylene-polyoxyethylene esters (PLURONIC®), andNP-40. In a specific aspect, the surfactant is selected from TWEEN(polysorbate) 20, TWEEN (polysorbate) 80 or a poloxamer. Theconcentration of the surfactant may range from about 0.001% to 1% (v:v)(preferably from about 0.001% to 0.1% (v:v), or 0.01% to 0.1% (v:v) suchas 0.001% (v:v), 0.005% (v:v), 0.01% (v:v), 0.02% (v:v), 0.05% (v:v),0.08% (v:v), 0.1% (v:v), 0.5% (v:v), or 1% (v:v) of the formulation,preferably 0.01% (v:v)). Throughout the present invention theconcentration of the surfactant will be given as % (v:v).

The formulation of the invention may also comprise one or more inorganicsalts. In one aspect, the concentration of inorganic salt should not bemore than 150 mM. Without being limiting, inorganic salts for use in theformulation of the invention can be selected from NaCl and KCl.Accordingly the formulation of the invention has an inorganic saltconcentration of 150 mM or lower, preferably 120 mM or lower, or 100 mMor lower, more preferably 90 mM or lower, 80 mM or lower, 75 mM orlower, such as 50 mM or lower or even 40 mM or lower, 25 mM or lower, 10mM or lower or 5 mM or lower. In one aspect, the formulation does notcontain any inorganic salt.

The polypeptides of the invention present in the formulation of theinvention should preferably have a melting temperature of at least 59°C. or more (such as 59.5° C. or more), preferably at least 60° C. ormore (such as 60.5° C. or more), more preferably at least 61° C. or more(such as 61.5° C. or more) or at least 62° C. or more (such as 62.5° C.or more), most preferably at least 63° C. or more (such as 63.5° C. ormore) as measured by the thermal shift assay (TSA) and/or differentialscanning calorimetry (DSC).

Without being limiting, melting point determination can be done by thefluorescence-based thermal shift assay which is based on the fact thatupon thermal unfolding the hydrophobic regions of proteins, usuallyhidden in the core of the protein fold, become accessible for binding toa hydrophobic fluorescent dye. The fluorescence emission of this dye isquenched in aqueous solution, whereas upon binding to the hydrophobicpatches of an unfolded protein a sharp increase in the fluorescenceyield of the probe is observed. Temperature induced unfolding istypically a two-state process with a sharp transition between the foldedand unfolded state, where the melting temperature (Tm) is defined as thetemperature at which half of the protein is in the unfolded state, i.e.the first derivative of the fluorescence signal upon gradual heating ofthe sample is plotted and the observed peak (or peaks when multipledomains and/or variants of the same domain are present) represents themelting temperature. The thermal shift assay can be performed in atypical real-time PCR instrument where melting curves can be recordedaccurately in high-throughput mode with only small quantities of proteinrequired.

During a differential scanning calorimetry experiment the sample isheated at a constant rate in an adiabatic environment (ΔT=0). The energyrequired to keep the temperature difference between a reference and thesample cell at zero is measured and yields the heat capacity as afunction of temperature (Cp(T)). The temperature corresponding to themaximum heat capacity represents the melting temperature (T_(m)). If thetemperature dependent unfolding process is reversible otherthermodynamic parameters such as the unfolding enthalpy (ΔH_(unfolding))can be determined.

Increased melting temperatures have been observed for the polypeptidesof the invention when present in a formulation that comprises anexcipient, preferably a saccharides and/or polyol such as mannitol,trehalose, sorbitol or sucrose. Accordingly, the present inventionrelates to a formulation comprising a polypeptide comprising one or moresingle variable domains, said formulation being formulated foradministration to a human subject, wherein said formulation furthercomprises at least an excipient, preferably a saccharide and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%); and wherein the melting temperature of thepolypeptide of the invention is at least 59° C. or more (such as 59.5°C. or more), preferably at least 60° C. or more (such as 60.5° C. ormore), more preferably at least 61° C. or more (such as 61.5° C. ormore) or at least 62° C. or more (such as 62.5° C. or more), mostpreferably at least 63° C. or more (such as 63.5° C. or more) asmeasured by the thermal shift assay (TSA) and/or differential scanningcalorimetry (DSC).

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier at a pH of 6.0 to 8.0 and a polypeptide comprisingone or more single variable domains, said formulation being formulatedfor administration to a human subject, wherein said formulation furthercomprises at least an excipient, preferably a saccharide and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%); wherein the melting temperature of the polypeptideof the invention is at least 59° C. or more (such as 59.5° C. or more),preferably at least 60° C. or more (such as 60.5° C. or more), morepreferably at least 61° C. or more (such as 61.5° C. or more) or atleast 62° C. or more (such as 62.5° C. or more), most preferably atleast 63° C. or more (such as 63.5° C. or more) as measured by thethermal shift assay (TSA) and/or differential scanning calorimetry(DSC).

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier at a pH of 6.0 to 8.0 and a polypeptide comprisingone or more single variable domains, said formulation being formulatedfor administration to a human subject, wherein said formulation furthercomprises at least an excipient, preferably a saccharide and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%), wherein said formulation has an inorganic saltconcentration of 150 mM or lower; and wherein the melting temperature ofthe polypeptide of the invention is at least 59° C. or more (such as59.5° C. or more), preferably at least 60° C. or more (such as 60.5° C.or more), more preferably at least 61° C. or more (such as 61.5° C. ormore) or at least 62° C. or more (such as 62.5° C. or more), mostpreferably at least 63° C. or more (such as 63.5° C. or more) asmeasured by the thermal shift assay (TSA) and/or differential scanningcalorimetry (DSC).

The formulation of the present invention exhibit stability when storedat a temperature of 37±5° C. The formulation of the invention mayexhibit stability when stored at a temperature of 37±5° C. for at least2 weeks, 3 weeks, 4 weeks, at least 5 weeks, at least 8 weeks, at least10 weeks, at least 3 months, at least 6 months, at least 1 year, 1.5year or even 2 years or more.

As is known to one skilled in the art, the temperatures indicated inthis text can be subject to normal variations.

Preferably, in those formulations that are stable under one or more ofthe above stress conditions:

-   -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms dimers (e.g. as assessed by        SE-HPLC) during storage under stress conditions, such as e.g. at        a temperature of 37±5° C. up to at least 2 weeks (preferably at        least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10        weeks, at least 3 months, at least 6 months, at least 1 year,        1.5 year or even 2 years or more); and/or    -   at least 80% (at least 85%, at least 90%, at least 95%, at least        98%, at least 99%, or at least 99.5%) of the polypeptide of the        invention retains its binding activity (e.g. as assessed by        ELISA and/or BIACORE) to at least one of its (preferably to all        of its) targets after storage under stress conditions, such as        e.g. at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more) compared to        the binding activity prior to the stress condition.

As indicated above, the polypeptides present in the formulation of theinvention preferably do not form dimers. The formation of dimers in thesample can e.g. be measured by SE-HPLC. For example, analysis in SE-HPLCof a formulation containing SEQ ID NO: 17 after storage for 10 weeks ata temperature of 37° C., showed the formation of a separate peak elutingat an apparent molecular weight of 44 kDa in comparison with molecularweight markers, while the monomeric polypeptide eluted between the 44and 17 kDa molecular weight markers. This separate peak at 44 kDarepresented a dimeric form of SEQ ID NO: 17. Preferably in theformulation of the invention, less than 10% (more preferably less than5%, even more preferably less than 3%, most preferably less than 1%) ofthe polypeptides forms dimers (e.g. as assessed by SE-HPLC) duringstorage under one or more of the above stress conditions.

Little to no dimer formation of the polypeptides of the invention hasbeen observed in formulations that comprise an excipient, preferably asaccharide and/or polyol such as mannitol, trehalose, sorbitol orsucrose. Accordingly, the present invention relates to a formulationcomprising a polypeptide comprising one or more single variable domains,said formulation being formulated for administration to a human subject,wherein said formulation further comprises at least an excipient,preferably a saccharide, a non-reducing sugar and/or polyol such asmannitol, trehalose, sorbitol or sucrose at a concentration of 1% to 20%(preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8%or 10%); wherein less than 10% (preferably less than 8%, more preferablyless than 7%, most preferably less than 5%) of the polypeptides formsdimers during one or more of the above stress conditions (such as duringstorage at a temperature of 37±5° C. up to at least 2 weeks (preferablyat least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks,at least 3 months, at least 6 months, at least 1 year, 1.5 year or even2 years or more)), the % of dimers as measured by SE-HPLC.

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastan excipient, preferably a saccharide, a non-reducing sugar and/orpolyol such as mannitol, trehalose, sorbitol or sucrose at aconcentration of 1% to 20% (preferably 2.5% to 15%, more preferably 5%to 10%, such as 5%, 7.5%, 8% or 10%); wherein less than 10% (preferablyless than 8%, more preferably less than 7%, most preferably less than5%) of the polypeptides forms dimers during one or more of the abovestress conditions (such as during storage at a temperature of 37±5° C.up to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,at least 8 weeks, at least 10 weeks, at least 3 months, at least 6months, at least 1 year, 1.5 year or even 2 years or more)), the % ofdimers as measured by SE-HPLC.

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastan excipient, preferably a saccharide, a non-reducing sugar and/orpolyol such as mannitol, trehalose, sorbitol or sucrose at aconcentration of 1% to 20% (preferably 2.5% to 15%, more preferably 5%to 10%, such as 5%, 7.5%, 8% or 10%); wherein said formulation has aninorganic salt concentration of 150 mM or lower; and wherein less than10% (preferably less than 8%, more preferably less than 7%, mostpreferably less than 5%) of the polypeptides forms dimers during one ormore of the above stress conditions (such as during storage at atemperature of 37±5° C. up to at least 2 weeks (preferably at least 3weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3months, at least 6 months, at least 1 year, 1.5 year or even 2 years ormore)), the % of dimers as measured by SE-HPLC.

Apart from this and/or in addition, the formulation of the presentinvention shows very little to no loss of potency and/or biologicalactivity of their polypeptides, even during storage under one or more ofthe above stress conditions.

The potency and/or biological activity of a biological describes thespecific ability or capacity of said biological to achieve a definedbiological effect. The potency and biological activities of thepolypeptides of the invention can be assessed by various assaysincluding any suitable in vitro assay, cell-based assay, in vivo assayand/or animal model known per se, or any combination thereof, dependingon the specific disease or disorder involved. Suitable in vitro assayswill be clear to the skilled person, and for example include ELISA; FACSbinding assay; BIACORE; competition binding assay (AlphaScreen®, PerkinElmer, Massachusetts, USA; FMAT); TRAP assay (osteoclast differentiationassay; Rissanen et al. 2005, J. Bone Miner. Res. 20, Suppl. 1: S256);NF-kappaB reporter gene assay (Mizukami et al. 2002, Mol. Cell. Biol.22: 992-1000). For example, SEQ ID NO: 17 interacts with RANKL andblocks the interaction of this ligand with RANK, thereby preventingsignalization through this receptor. SEQ ID NO's: 18 to 20 interact withIL-6R and block the interaction of this receptor with IL-6. The potencyof the polypeptides of the invention for blocking the respectiveligand/receptor interaction can be determined, e.g. by ELISA, BIACORE,AlphaScreen®.

For example, in one embodiment, BIACORE kinetic analysis uses SurfacePlasmon Resonance (SPR) technology to monitor macromolecularinteractions in real time and is used to determine the binding on andoff rates of polypeptides of the formulation of the invention to theirtarget. BIACORE kinetic analysis comprises analyzing the binding anddissociation of the target from chips with immobilized polypeptides ofthe invention on their surface. A typical BIACORE kinetic study involvesthe injection of 250 μL of polypeptide reagent at varying concentrationin HBS buffer containing 0.005% TWEEN (polysorbate) 20 over a sensorchip surface, onto which has been immobilized the antigen. In theBIACORE 3000 system, the ligand is immobilized on carboxymethylateddextran over a gold surface, while the second partner (analyte) iscaptured as it flows over the immobilized ligand surface. Theimmobilized ligands are remarkably resilient and maintain theirbiological activity. The bound analytes can be stripped from theimmobilized ligand without affecting its activity to allow many cyclesof binding and regeneration on the same immobilized surface. Interactionis detected in real time via SPR and at high sensitivity. Because thesame affinity may reflect different on-rates and off-rates, thisinstrument excels over most other affinity measuring methods in that itmeasures on-rates (ka) and off-rates (kd). Concentration determinationexperiments are also feasible.

The formulation of the present invention exhibits almost no loss inbiological activities of the polypeptide during the prolonged storageunder the conditions described above, as assessed by variousimmunological assays including, for example, enzyme-linked immunosorbentassay (ELISA) and Surface Plasmon Resonance to measure the ability ofthe polypeptide to specifically bind to an antigen. The polypeptidespresent in the formulation of the present invention retain, even underthe above defined stress conditions (such as storage under certaintemperature stress for defined periods) more than 80%, more than 85%,more than 90%, more than 95%, more than 98%, more than 99%, or more than99.5% of their initial biological activities (e.g., the ability to bindto vWF, RANKL, IL-6R and/or HSA) of the polypeptides prior to thestorage. In some embodiments, the polypeptides in the formulation of theinvention retain under the above defined stress conditions at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, orat least 99.5% of the biological activity (e.g., the ability to bind tovWF, RANKL, IL-6R and/or HSA) compared to the polypeptides present in areference formulation prior to the storage.

In one embodiment, the polypeptides of the invention bind HSA. In theformulations of the present invention, at least 80% (preferably at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least99.5%) of said polypeptides retain their binding activity to HSA underone or more of the above stress conditions (such as storage undercertain temperature stress for defined periods) compared to the bindingactivity prior to the stress condition. Without being limiting, thebinding of the polypeptides to HSA can be determined e.g. by ELISAand/or BIACORE.

In a preferred aspect, at least 80% (at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5%) of thepolypeptides present in the formulation of the invention retain theirbinding activity to all of their targets (such as e.g. RANKL and HSA,IL-6R and HSA or IL-23 and HSA) after storage under one or more of theabove stress conditions compared to the binding activity prior tostorage.

Suitable animal models for determining the potency and/or biologicalactivity of the polypeptides present in the formulations of theinvention will be clear to the skilled person and will depend on theintended disease and/or disorder to be prevented and/or treated.Suitable animal models for testing the potency and/or biologicalactivity of the polypeptides of the invention are e.g. described in WO08/020079, WO 09/068627 and WO 08/142164.

Little to no loss of potency of the polypeptides of the invention hasbeen observed in formulations that comprise an excipient, preferably asaccharide, non-reducing sugar and/or polyol such as mannitol, sorbitol,trehalose or sucrose. Accordingly, the present invention relates to aformulation comprising a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastan excipient, preferably a saccharide, non-reducing sugar and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose, at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%); wherein at least 80% (preferably at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)of the polypeptides retain their binding activity to at least one(preferably to all) of their targets under one or more of the abovestress conditions (such as during storage at a temperature of 37±5° C.up to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,at least 8 weeks, at least 10 weeks, at least 3 months, at least 6months, at least 1 year, 1.5 year or even 2 years or more)) compared tothe binding activity prior to the stress conditions, said bindingactivity as measured by ELISA and/or BIACORE.

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastan excipient, preferably a saccharide, non-reducing sugar and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose, at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%); wherein at least 80% (preferably at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)of the polypeptides retain their binding activity to at least one(preferably to all) of their targets under one or more of the abovestress conditions (such as during storage at a temperature of 37±5° C.up to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,at least 8 weeks, at least 10 weeks, at least 3 months, at least 6months, at least 1 year, 1.5 year or even 2 years or more)) compared tothe binding activity prior to the stress conditions, said bindingactivity as measured by ELISA and/or BIACORE.

Accordingly, the present invention relates to a formulation comprisingan aqueous carrier and a polypeptide comprising one or more singlevariable domains, said formulation being formulated for administrationto a human subject, wherein said formulation further comprises at leastan excipient, preferably a saccharide, non-reducing sugar and/or polyolsuch as mannitol, sorbitol, trehalose or sucrose, at a concentration of1% to 20% (preferably 2.5% to 15%, more preferably 5% to 10%, such as5%, 7.5%, 8% or 10%); wherein said formulation has an inorganic saltconcentration of 150 mM or lower; and wherein at least 80% (preferablyat least 85%, at least 90%, at least 95%, at least 98%, at least 99%, orat least 99.5%) of the polypeptides retain their binding activity to atleast one (preferably to all) of their targets under one or more of theabove stress conditions (such as during storage at a temperature of37±5° C. up to at least 2 weeks (preferably at least 3 weeks, at least 5weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at least6 months, at least 1 year, 1.5 year or even 2 years or more)) comparedto the binding activity prior to the stress conditions, said bindingactivity as measured by ELISA and/or BIACORE.

Accordingly, in the stable formulations of the present inventionpreferably:

-   -   the polypeptide of the invention has a melting temperature of at        least 59° C. or more (such as 59.5° C. or more), preferably at        least 60° C. or more (such as 60.5° C. or more), more preferably        at least 61° C. or more (such as 61.5° C. or more) or at least        62° C. or more (such as 62.5° C. or more), most preferably at        least 63° C. or more (such as 63.5° C. or more) (e.g. as        assessed by TSA or DSC);    -   less than 10% (more preferably less than 5%, even more        preferably less than 3%, most preferably less than 1%) of the        polypeptide of the invention forms dimers (e.g. as assessed by        SE-HPLC) during storage under one or more stress conditions,        such as e.g. at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more); and/or    -   at least 80% (at least 85%, at least 90%, at least 95%, at least        98%, at least 99%, or at least 99.5%) of the polypeptide of the        invention retains its binding activity (e.g. as assessed by        ELISA and/or BIACORE) to at least one (preferably to all) of its        targets after storage under one or more stress conditions, such        as e.g. at a temperature of 37±5° C. up to at least 2 weeks        (preferably at least 3 weeks, at least 5 weeks, at least 8        weeks, at least 10 weeks, at least 3 months, at least 6 months,        at least 1 year, 1.5 year or even 2 years or more) compared to        the binding activity prior to the stress condition.

General methods for producing the single variable domains and/orpolypeptides present in the formulation of the invention are known tothe skilled person and/or have been described in the art. The singlevariable domains and/or polypeptides can be produced in any host knownto the skilled person. For example but without being limiting, thesingle variable domains and/or polypeptides can be produced inprokaryotic hosts among which E. coli or eukaryotic hosts, for exampleeukaryotic host selected from insect cells, mammalian cells, and lowereukaryotic hosts comprising yeasts such as Pichia, Hansenula,Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora,Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, preferably Pichia pastoris. Production ofNanobodies in prokaryotes and lower eukaryotic hosts such as Pichiapastoris has e.g. been described in WO 94/04678, WO 94/25591 and WO08/142164. The contents of these applications are explicitly referred toin the connection with general culturing techniques and methods,including suitable media and conditions. The contents of these documentsare incorporated by reference. The skilled person can also devisesuitable genetic constructs for expression of the polypeptides of theinvention in different hosts on the basis of the present application andcommon general knowledge. The present invention also relates toconditions and genetic constructs described in the art, for example thegeneral culturing methods, plasmids, promoters and leader sequencesdescribed in WO 94/25591, WO 08/020079, Gasser et al. 2006 (Biotechnol.Bioeng. 94: 535); Gasser et al. 2007 (Appl. Environ. Microbiol. 73:6499); or Damasceno et al. 2007 (Microbiol. Biotechnol. 74: 381).

More particularly, the method for the expression and/or production of apolypeptide comprising one or more single variable domains at leastcomprising the steps of:

-   -   d) cultivating a host or host cell (as defined herein) under        conditions that are such that said host or host cell will        multiply;    -   e) maintaining said host or host cell under conditions that are        such that said host or host cell expresses and/or produces the        polypeptide;    -   f) isolating and/or purifying the secreted polypeptide from the        medium.

To produce/obtain expression of the polypeptide, the transformed hostcell or transformed host organism may generally be kept, maintainedand/or cultured under conditions such that the (desired) polypeptide isexpressed/produced. Suitable conditions will be clear to the skilledperson and will usually depend upon the host cell/host organism used, aswell as on the regulatory elements that control the expression of the(relevant) nucleotide sequence. Again, reference is made to thehandbooks and patent applications mentioned above.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

The polypeptide of the invention may then be isolated from the hostcell/host organism and/or from the medium in which said host cell orhost organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the polypeptide of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the polypeptide to be isolated).

In the present invention, the host can be removed from the culturemedium by routine means. For example, the host can be removed bycentrifugation or filtration. The solution obtained by removal of thehost from the culture medium is also referred to as culture supernatant,or clarified culture supernatant. The polypeptides of the invention canbe purified from the culture supernatant by standard methods. Standardmethods include, but are not limited to chromatographic methods,including size exclusion chromatography, hydrophobic interactionchromatography, ion exchange chromatography, and affinitychromatography. These methods can be performed alone or in combinationwith other purification methods, e.g. precipitation or gelelectrophoresis. The skilled person can devise suitable combinations ofpurification methods for the polypeptides of the invention on the basisof common general knowledge. For specific examples the art cited hereinis referred to.

In one exemplary embodiment, the polypeptides of the invention can bepurified from culture supernatant by a combination of affinitychromatography on Protein A, ion exchange chromatography and sizeexclusion chromatography. Reference to any “step of purification”,includes, but is not limited to these particular methods.

More specifically, the polypeptides of the invention can be purifiedfrom culture supernatant using a process wherein the clarifiedsupernatant (obtained by centrifugation) is captured on any combinationof columns selected from (without being limiting) affinitychromatography resin such as Protein A resin, Cation ExchangeChromatography (CIEC) or an Anion Exchange Chromatography (AIEC) usingfor example Poros 50HS (POROS), SOURCE 305 or SOURCE 155 (GEHealthcare), SP SEPHAROSE (GE Healthcare), CAPTO S (GE Healthcare),CAPTO MMC (GE Healthcare) or Poros 50HQ (POROS), SOURCE 30Q or SOURCE15Q (GE Healthcare), Q SEPHAROSE (GE Healthcare), CAPTO Q and DEAESEPHAROSE e (GE Healthcare), Size exclusion chromatography (SE-HPLC)using for example SUPERDEX 75 or SUPERDEX 200 (GE Healthcare),hydrophobic interaction chromatography (HIC) using for example octyl,butyl SEPHAROSE or equivalents, optionally also including a tangentialflow filtration (TFF) step. Any combination of columns can be used forthe purification of the polypeptides of the invention, such as e.g.Protein A resin followed by Cation Exchange Chromatography or two CationExchange Chromatography steps.

The present invention also provides methods for preparing the stableformulations of the invention comprising the polypeptides of theinvention. More particularly, the present invention provides methods forpreparing stable formulations of such polypeptides, said methodscomprising concentrating a fraction containing the purified polypeptideto the final polypeptide concentration using e.g. a semipermeablemembrane with an appropriate molecular weight (MW) cutoff (e.g. a 5 kDcutoff for single variable domains; a 10 kD cutoff for bivalentpolypeptides comprising two single variable domains; or a 15 kD cutofffor trivalent polypeptides comprising three single variable domains) anddiafiltering and/or ultrafiltering to buffer exchange and furtherconcentrate the polypeptide fraction into the formulation buffer usingthe same membrane. As extensively described above, the formulationbuffer of the present invention may further comprise an excipient at aconcentration of 1% to 20%.

The pH of the formulation may range from about 5.5 to about 8.0, or mayrange from about 6.0 to about 7.5, preferably from about 6.2 to 7.5,from about 6.2 to 7.0, most preferably from about 6.5 to 7.0.

Surfactant (e.g. TWEEN (polysorbate) 20, TWEEN (polysorbate) 80 orpoloxamer) may be added after the final diafiltration/ultrafiltrationstep at a concentration in the range of about 0% to 1%, preferably0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%,0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or0.005%.

The formulation of the present invention may be sterilized by varioussterilization methods, including sterile filtration, radiation, etc. Ina specific embodiment, the polypeptide formulation is filter-sterilizedwith a presterilized 0.2 micron filter.

Preferably, the formulation of the present invention is supplied in ahermetically sealed container. Liquid formulations may comprise aquantity between 1 mL and 20 mL, preferably about 1 mL, 2 mL, 3 mL, 4mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20 mL.

The formulation of the present invention can be prepared as unit dosageforms by preparing a vial containing an aliquot of the formulation for aone time use. For example, a unit dosage of liquid formulation per vialmay contain 1 mL, 2 mL, 3 mL, 4 mL, mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL,15 mL, or 20 mL of the formulation. The pharmaceutical unit dosage formscan be made suitable for any form of delivery of the polypeptide of theinvention including (without being limiting) parenteral delivery,topical delivery, pulmonary delivery, intranasal delivery, vaginaldelivery, enteral delivery, rectal delivery, oral delivery and/orsublingual delivery. In one aspect, the present invention relates to apharmaceutical unit dosage form suitable for parenteral (such as e.g.intravenous, intraarterial, intramuscular, intracerebral, intraosseous,intradermal, intrathecal, intraperitoneal, subcutaneous, etc)administration to a subject, comprising a formulation of the inventionin a suitable container. In another preferred aspect, the subject is ahuman. In another specific embodiment, the formulations of the presentinvention are formulated into single dose vials as a sterile liquid thatcontains 10 mg/mL of one of SEQ ID NO's: 7 to 12, 10 mM histidine bufferat pH 6.0, 10% sucrose and 0.0005% TWEEN (polysorbate) 80.

The amount of a formulation of the present invention which will beeffective in the prevention, treatment and/or management of a certaindisease or disorder can be determined by standard clinical techniqueswell-known in the art or described herein. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. For formulations of the polypeptide, encompassed bythe invention, the dosage administered to a patient may further becalculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg.

The required volume (in mL) to be given is then determined by taking themg dose required divided by the concentration of the polypeptideformulation. The final calculated required volume will be obtained bypooling the contents of as many vials as are necessary into syringe(s)to administer the polypeptide formulation of the invention.

The present invention also encompasses a finished packaged and labelledpharmaceutical product. This article of manufacture or kit includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In oneembodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses formulations, preferably sterile, suitable foreach delivery route. In the case of dosage forms suitable for parenteraladministration (such as e.g. subcutaneous administration) the activeingredient, e.g., polypeptide of the invention, is sterile and suitablefor administration as a particulate free solution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises the formulation containing the polypeptide. Thepackaging material includes instruction means which indicate that saidpolypeptide can be used to prevent, treat and/or manage one or moresymptoms associated with the disease or disorder by administeringspecific doses and using specific dosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a formulation containing the polypeptide of interest, andwherein said packaging material includes instruction means whichindicate that said agents can be used to prevent, treat and/or managethe disease or disorder by administering specific doses and usingspecific dosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a formulation containing the polypeptide, and wherein saidpackaging material includes instruction means which indicate that saidagents can be used to prevent, treat and/or manage one or more symptomsassociated with the disease or disorder by administering specific dosesand using specific dosing regimens as described herein.

The formulations, containers, pharmaceutical unit dosages and kits ofthe present invention may be administered to a subject to prevent, treatand/or manage a specific disease and/or disorder. In a specific aspect,the formulations, containers, pharmaceutical unit dosages and kits ofthe present invention are administered to a subject to prevent, treatand/or manage a disease and/or disorder associated with or characterizedby aberrant expression and/or activity of a certain target or one ormore symptoms thereof. In another specific aspect, the formulations,containers, pharmaceutical unit dosages and kits of the presentinvention are administered to a subject to prevent, treat and/or managediseases and/or disorders associated with aberrant expression and/oractivity of RANKL, diseases and/or disorders associated withoverexpression of IL-6, or vascular diseases and/or disorders or one ormore symptoms thereof.

Diseases and disorders associated with aberrant expression and/oractivity of RANKL are for example bone diseases and disorders, andinclude (without being limiting) the following diseases and disorders:Osteoporosis (McClung 2006, Current Osteoporosis Reports 4: 28-33),including, but not limited to, primary osteoporosis, endocrineosteoporosis (including, but not limited to, hyperthyroidism,hyperparathyroidism (Anandarajah and Schwarz 2006, J. Cell Biochem. 97:226-232), Cushing's syndrome, and acromegaly), hereditary and congenitalforms of osteoporosis (including, but not limited to, osteogenesisimperfecta, homocystinuria, Menkes' syndrome, Riley-Day syndrome),osteoporosis due to immobilization of extremities,glucocorticoid-induced osteoporosis (Locklin et al. 2001, Bone 28(Suppl.): 580; McClung 2006, Current Osteoporosis Reports 4: 28-33;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232) andpost-menopausal osteoporosis (McClung 2006, Current Osteoporosis Reports4: 28-33); (Juvenile or Familial) Paget's disease (Cundy et al. 2002,Hum. Mol. Genet. 11: 2119-2127; Whyte et al. 2002, J. Bone Miner. Res.17: 26-29; Whyte et al. 2002, N. Engl. J. Med. 347: 175-184;Johnson-Pais et al. 2003, J. Bone Miner Res. 18: 376-380; Anandarajahand Schwarz 2006, J. Cell Biochem. 97: 226-232; Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232); Osteomyelitis, i.e., an infectiouslesion in bone, leading to bone loss; Hypercalcemia (Anandarajah andSchwarz 2006, J. Cell Biochem. 97: 226-232), including, but not limitedto, hypercalcemia resulting from solid tumors (including, but notlimited to, breast, lung and kidney) and hematologic malignancies(including, but not limited to, multiple myeloma (Sordillo and Pearse2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003, CancerRes. 63: 287-289), lymphoma and leukemia), idiopathic hypercalcemia, andhypercalcemia associated with hyperthyroidism and renal functiondisorders; Bone loss, including but not limited to, osteopenia followingsurgery, osteopenia induced by steroid administration, osteopeniaassociated with disorders of the small and large intestine, andosteopenia associated with chronic hepatic and renal diseases;Osteonecrosis, i.e., bone cell death, including, but not limited to,osteonecrosis associated with traumatic injury, osteonecrosis associatedwith Gaucher's disease, osteonecrosis associated with sickle cellanemia, osteonecrosis associated with systemic lupus erythematosus,osteonecrosis associated with rheumatoid arthritis, osteonecrosisassociated with periodontal disease, osteonecrosis associated withosteolytic metastasis, and osteonecrosis associated with othercondition; Bone loss associated with arthritic disorders such aspsoriatic arthritis, rheumatoid arthritis, loss of cartilage and jointerosion associated with rheumatoid arthritis (Bezerra et al. 2005,Brazilian Journal of Medical and Biological Research 38: 161-170;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170), including inflammatory arthritis (McClung 2006,Current Osteoporosis Reports 4: 28-33), Collagen-induced arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170); Periprosthetic osteolysis (McClung 2006, CurrentOsteoporosis Reports 4: 28-33; Anandarajah and Schwarz 2006, J. CellBiochem. 97: 226-232); Cancer-related bone disease (McClung 2006,Current Osteoporosis Reports 4: 28-33); Bone loss associated witharomatase inhibitor therapy (Lewiecki 2006, Expert Opin. Biol. Ther. 6:1041-1050); Bone loss associated with androgen deprivation therapy(Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050); Bone lossassociated bone metastasis; Bone loss associated with diseases havingimmune system involvement, such as adult and childhood leukaemias,cancer metastasis, autoimmunity, and various viral infections (HolsteadJones et al. 2002, Ann. Rheum. Dis. 61 (Suppl II): ii32-ii39) Osteopenicdisorders such as adult and childhood leukaemia (Oliveri et al. 1999,Henry Ford Hosp. Med. 39: 45-48), chronic infections such as hepatitis Cor HIV (Stellon et al. 1985, Gastroenterology 89: 1078-1083), autoimmunedisorders such as diabetes mellitus (Piepkorn et al. 1997, Horm. Metab.Res. 29: 584-91), and lupus erythematosus (Seitz et al. 1985, Ann. RheumDis. 44: 438-445), allergic diseases such as asthma (Ebeling et al.1998, J. Bone Min. Res. 13: 1283-1289), lytic bone metastases inmultiple cancers such as breast cancer (Coleman 1998, Curr. Opin. Oncol.10 (Suppl 1): 7-13); Prostate cancer; Myeloma bone disease (Anandarajahand Schwarz 2006, J. Cell Biochem. 97: 226-232); Periodontal infections(Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Expansileskeletal hyperphosphatasia (Anandarajah and Schwarz 2006, J. CellBiochem. 97: 226-232); Bone metastases (Lewiecki 2006, Expert Opin.Biol. Ther. 6: 1041-1050; Anandarajah and Schwarz 2006, J. Cell Biochem.97: 226-232).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the formulations, containers,pharmaceutical unit dosages and kits of the invention of other diseasesand disorders associated with an imbalance in the RANKL/RANK/OPGpathway. Such diseases and disorders include but are not limited toosteoporosis, inflammatory conditions, autoimmune conditions, asthma,rheumatoid arthritis, multiple sclerosis, Multiple myeloma (Sordillo andPearse 2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003,Cancer Res. 63: 287-289); Vascular diseases (Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232) and Cardiovascular disease (Lewiecki2006, Expert Opin. Biol. Ther. 6: 1041-1050).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the formulations, containers,pharmaceutical unit dosages and kits of the invention of diseases anddisorders associated with osteopetrosis such as osteopetrosis tarda,osteopetrosis congenita and marble bone disease.

Disease and disorders caused by excessive IL-6 production include sepsis(Starnes et al., 1999) and various forms of cancer such as multiplemyeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia(Klein et al., 1991), lymphoma, B-lymphoproliferative disorder (BLPD)and prostate cancer. Non-limiting examples of other diseases caused byexcessive IL-6 production or signalling include bone resorption(osteoporosis) (Roodman et al., 1992; Jilka et al., 1992), cachexia(Strassman et al., 1992), psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie etal., 1994), inflammatory diseases and disorder such as rheumatoidarthritis, systemic onset juvenile idiopathic arthritis,hypergammaglobulinemia (Grau et al., 1990); Crohn's disease, ulcerativecolitis, systemic lupus erythematosus (SLE), multiple sclerosis,Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (inparticular allergic asthma) and autoimmune insulin-dependent diabetesmellitus (Campbell et al., 1991).

Vascular diseases and/or disorders include acute coronary syndrome(ACS), myocardial infarction, thrombotic thrombocytopenic purpura (TTP)or Moschcowitz syndrome, vascular surgery and stroke.

The formulations, containers, pharmaceutical unit dosages and kits ofthe present invention may also be advantageously utilized in combinationwith one or more other therapies (e.g., one or more other prophylacticor therapeutic agents), preferably therapies useful in the prevention,treatment and/or management of the (same or another) disease ordisorder. When one or more other therapies (e.g., prophylactic ortherapeutic agents) are used, they can be administered separately, inany appropriate form and by any suitable route. Therapeutic orprophylactic agents include, but are not limited to, small molecules,synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g.,DNA and RNA nucleotides including, but not limited to, antisensenucleotide sequences, triple helices, RNAi, and nucleotide sequencesencoding biologically active proteins, polypeptides or peptides),antibodies, other single variable domains, synthetic or naturalinorganic molecules, mimetic agents, and synthetic or natural organicmolecules. Any therapy (e.g., prophylactic or therapeutic agents) whichis known to be useful, or which has been used or is currently being usedfor the prevention, treatment and/or management of one or more symptomsassociated with a specific disease or disorder, can be used incombination with the formulations of the present invention in accordancewith the invention described herein.

A formulation of the invention may be administered to a mammal,preferably a human, concurrently with one or more other therapies (e.g.,one or more other prophylactic or therapeutic agents). The term“concurrently” is not limited to the administration of prophylactic ortherapeutic agents/therapies at exactly the same time, but rather it ismeant that the formulation of the invention and the other agent/therapyare administered to a mammal in a sequence and within a time intervalsuch that the polypeptide contained in the formulation can act togetherwith the other agent/therapy to provide an increased benefit than ifthey were administered otherwise. For example, the formulation of theinvention and the one or more other prophylactic or therapeutic agentsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic or prophylactic effect.

When used in combination with other therapies (e.g., prophylactic and/ortherapeutic agents), the formulations of the invention and the othertherapy can act additively or synergistically. The inventioncontemplates administration of a formulation of the invention incombination with other therapies (e.g., prophylactic or therapeuticagents) by the same or different routes of administration, e.g., oraland parenteral.

Various delivery systems are known and can be used to administer theformulation of the present invention. Methods of administeringformulations of the present invention include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and, preferably subcutaneous), epiduraladministration, topical administration, and mucosal administration(e.g., intranasal and oral routes). In a specific embodiment, liquidformulations of the present invention are administered parenteral.

A particular advantage of the NFDs described in this invention is theability to assemble functionally or partly functionally during e.g. themanufacturing process (e.g. purification step etc) in a controllablemanner. A dimerization principle is used which allows the formation ofhomodimers. Examples described herein include NFDs-Mo, NFDs-Di, andNFDs-Tri. In these cases, the monomeric building blocks are expressed ina bacterial system and then bound in high concentration to a separationchromatographic device, e.g. Protein A or IMAC, and eluted swiftly toretain the desired dimeric complexes, i.e. the NFDs, in substantialyield. Under these conditions, the homodimeric proteins form bythemselves and can directly be isolated in the dimeric form by saidseparation step and/or further isolated by size exclusionchromatography.

The present invention is further illustrated by the following preferredaspects and examples, which in no way should be construed as furtherlimiting. The entire contents of all of the references (includingliterature references, issued patents, published patent applications,and co-pending patent applications) cited throughout this applicationare hereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

Preferred Aspects

-   1. A formulation, such as a pharmaceutical formulation, comprising    an aqueous carrier having a pH of 5.5 to 8.0 and a polypeptide    comprising one or more single variable domains at a concentration of    1 mg/mL to 200 mg/mL, said formulation being formulated for    administration to a human subject and said formulation further    comprising one or more components selected from:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%;-   c) A surfactant at a concentration of 0.001% to 1% selected from    TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer,    -   wherein said formulation has an inorganic salt concentration of        150 mM or lower.-   2. The formulation of aspect 1, wherein the inorganic salt    concentration is from 50 mM to 100 mM or lower.-   3. The formulation of aspect 2, that does not contain any inorganic    salt.-   4. The formulation of any of aspects 1 to 3, wherein the formulation    comprises a buffer at a concentration of 10 mM to 100 mM selected    from the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0,    MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   5. The formulation of aspect 4, wherein the formulation comprises a    histidine buffer pH 6.5.-   6. The formulation of aspect 4, wherein the formulation comprises a    histidine buffer pH 6.0.-   7. The formulation of any of aspects 4 to 6, wherein the buffer has    a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as 10    mM or 15 mM.-   8. The formulation of any of aspects 1 to 7, wherein the formulation    comprises a surfactant at a concentration of 0.001% to 1% selected    from TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.-   9. The formulation of aspect 8, wherein the formulation comprises    TWEEN (polysorbate) 80.-   10. The formulation of any of aspects 8 or 9, wherein the surfactant    has a concentration of 0.001% to about 0.1%, or about 0.01% to about    0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%,    or 1% of the formulation, preferably 0.01%.-   11. The formulation of any of aspects 1 to 3, wherein the    formulation comprises an excipient at a concentration of 1% to 20%.-   12. The formulation of aspect 11, wherein the excipient is a    dissaccharide and/or a polyol.-   13. The formulation of aspect 11, wherein the excipient is selected    from sucrose, mannitol, sorbitol and trehalose.-   14. The formulation of any of aspects 11 to 13, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   15. The formulation of any of aspects 1 to 14, wherein the    polypeptide has a solubility of at least 20 mg/mL as determined by    the PEG exclusion method or by centrifugal ultrafiltration.-   16. The formulation of aspect 15, wherein the polypeptide has a    solubility of at least 50 mg/mL as determined by the PEG exclusion    method or by centrifugal ultrafiltration.-   17. The formulation of aspect 16, wherein the polypeptide has a    solubility of at least 90 mg/mL as determined by the PEG exclusion    method or by centrifugal ultrafiltration.-   18. The formulation of aspect 17, wherein the polypeptide has a    solubility of at least 120 mg/mL as determined by the PEG exclusion    method or by centrifugal ultrafiltration.-   19. The formulation of aspect 18, wherein the polypeptide has a    solubility of at least 150 mg/mL as determined by the PEG exclusion    method or by centrifugal ultrafiltration.-   20. The formulation of aspect 19, wherein the polypeptide has a    solubility of at least 200 mg/mL as determined by the PEG exclusion    method or by a concentration experiment.-   21. The formulation of any of aspects 1 to 20, wherein the    formulation at least comprises one or more components selected from:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   c) A surfactant at a concentration of 0.001% to 1% selected from    TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.-   22. The formulation of aspect 21, wherein the formulation comprises    a buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   23. The formulation of aspect 22, wherein the formulation comprises    a histidine buffer pH 6.5.-   24. The formulation of aspect 22, wherein the formulation comprises    a histidine buffer pH 6.0.-   25. The formulation of any of aspects 22 to 24, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   26. The formulation of any of aspects 21 to 25, wherein the    formulation comprises a surfactant at a concentration of 0.001% to    1% selected from TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a    poloxamer.-   27. The formulation of aspect 26, wherein the formulation comprises    TWEEN (polysorbate) 80.-   28. The formulation of any of aspects 26 or 27, wherein the    surfactant has a concentration of 0.001% to about 0.1%, or about    0.01% to about 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%,    0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01%.-   29. The formulation of any of aspect 1 to 28, wherein the    polypeptide has a melting temperature of at least 59° C. or more    (such as 59.5° C. or more), preferably at least 60° C. or more (such    as 60.5° C. or more), more preferably at least 61° C. or more (such    as 61.5° C. or more) or at least 62° C. or more (such as 62.5° C. or    more), most preferably at least 63° C. or more (such as 63.5° C. or    more) as measured by the thermal shift assay (TSA) and/or    differential scanning calorimetry (DSC).-   30. The formulation of aspect 29, which has a pH of 6.2 to 7.5.-   31. The formulation of aspect 30, which has a pH of 6.5 to 7.5.-   32. The formulation of aspect 31, which has a pH of 6.5 to 7.0.-   33. The formulation of any of aspects 29 to 32, wherein the    formulation at least comprises one or more components.-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES    pH6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%.-   34. The formulation of aspect 33, wherein the formulation comprises    a buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   35. The formulation of aspect 34, wherein the formulation comprises    a histidine buffer pH 6.0-6.5 or a hepes buffer pH 7.0.-   36. The formulation of any of aspects 34 or 35, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   37. The formulation of any of aspects 33 to 36, wherein the    formulation comprises an excipient at a concentration of 1% to 20%.-   38. The formulation of aspect 37, wherein the excipient is a    dissaccharide and/or a polyol.-   39. The formulation of aspect 38, wherein the excipient is selected    from sucrose, mannitol, sorbitol and trehalose.-   40. The formulation of any of aspects 37 to 39, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   41. The formulation of any of aspects 1 to 40, wherein the    polypeptide is stable after multiple (up to 10) freeze/thaw cycles,    said stability as determined by SE-HPLC, IEX-HPLC, RP-HPLC, BIACORE    analysis and/or potency assay.-   42. The formulation of any of aspects 1 to 41, wherein the    polypeptide is stable during storage at a temperature of 2-8° C. up    to up to at least 2 weeks (preferably at least 3 weeks, at least 5    weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at    least 6 months, at least 1 year, 1.5 year or even 2 years or more),    said stability as determined by OD320/OD280 measurement, elastic    light scattering, SE-HPLC and/or RP-HPLC.-   43. The formulation of aspects 42, wherein no particulates are    present as measured by OD320/OD280 measurement and/or elastic light    scattering.-   44. The formulation of any of aspects 42 or 43, in which the    OD320/OD280 is 0.05 or less.-   45. The formulation of any of aspects 42 or 43, in which the    scattering in elastic light scattering stays within the detection    range, and/or preferably is 1000 abs or less.-   46. The formulation of any of aspects 42 to 45, wherein the    formulation comprises a buffer at a concentration of 10 mM to 100 mM    selected from the group consisting of histidine pH 6.0-6.5, hepes pH    7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   47. The formulation of aspect 46, wherein the formulation comprises    a histidine buffer pH 6.0-6.5 or a hepes buffer pH 7.0.-   48. The formulation of any of aspects 46 or 47, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   49. The formulation of any of aspects 1 to 48, wherein the    polypeptide is stable during storage at a temperature of 37±5° C. up    to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,    at least 8 weeks, at least 10 weeks, at least 3 months, at least 6    months, at least 1 year, 1.5 year or even 2 years or more), said    stability as determined by OD320/OD280 measurement, elastic light    scattering, SE-HPLC, RP-HPLC, IEX-HPLC, potency assay (such as    BIACORE or ELISA) and/or SDS-PAGE.-   50. The formulation of aspect 49, wherein less than 10% (preferably    less than 8%, more preferably less than 7%, most preferably less    than 5%) of the polypeptides forms pyroglutamate at the N-terminal    glutamic acid during storage, the % of pyroglutamate as measured by    RP-HPLC.-   51. The formulation of aspect 50, which has a pH of 7.0 or less.-   52. The formulation of any of aspects 50 or 51, wherein the    formulation comprises a buffer at a concentration of 10 mM to 100 mM    selected from the group consisting of histidine pH 6.0-6.5, hepes pH    7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   53. The formulation of aspect 52, wherein the formulation comprises    a histidine buffer or an acetate buffer.-   54. The formulation of any of aspects 52 or 53, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   55. The formulation of any of aspects 49 to 54, wherein less than    10% (preferably less than 7.5%, more preferably less than 5%, most    preferably less than 2%) of the polypeptides forms dimers during    storage, the % of dimers as measured by SE-HPLC.-   56. The formulation of aspect 55, wherein the formulation at least    comprises one or more components selected from:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%.-   57. The formulation of aspect 56, wherein the formulation comprises    a buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   58. The formulation of aspect 57, wherein the formulation comprises    a histidine buffer or an acetate buffer.-   59. The formulation of any of aspects 57 or 58, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   60. The formulation of any of aspects 55 to 59, wherein the    formulation comprises an excipient at a concentration of 1% to 20%.-   61. The formulation of aspect 60, wherein the excipient is a    disaccharide and/or a polyol.-   62. The formulation of aspect 60, wherein the excipient is a    non-reducing sugar.-   63. The formulation of aspect 61 or 62, wherein the excipient is    selected from trehalose, mannitol and sucrose.-   64. The formulation of any of aspects 60 to 63, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   65. The formulation of any of aspects 49 to 64, wherein no    particulates are present as measured by OD320/OD280 measurement    and/or elastic light scattering.-   66. The formulation of aspect 65, in which the OD320/OD280 is 0.05    or less.-   67. The formulation of aspect 65, in which the scattering in elastic    light scattering stays within the detection range, and/or preferably    is 1000 abs or less.-   68. The formulation of any of aspects 65 to 67, wherein the    formulation comprises a buffer at a concentration of 10 mM to 100 mM    selected from the group consisting of histidine pH 6.0-6.5, hepes pH    7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   69. The formulation of aspect 68, wherein the formulation comprises    a histidine buffer.-   70. The formulation of any of aspects 68 or 69, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   71. The formulation of any of aspects 49 to 70, wherein at least 80%    (preferably at least 90%, more preferably at least 95% or even at    least 99%) of the polypeptides retain their binding activity to at    least one of their targets after storage compared to the binding    activity prior to storage, said binding activity as measured by    ELISA and/or BIACORE.-   72. The formulation of aspect 71, wherein the formulation at least    comprises one or more components selected from:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of hepes pH 7.0-8.0, histidine pH 6.0-6.5, MES pH    6.0 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%.-   73. The formulation of aspect 72, wherein the formulation comprises    a buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0.-   74. The formulation of aspect 73, wherein the formulation comprises    a histidine buffer.-   75. The formulation of any of aspects 73 or 74, wherein the buffer    has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as    10 mM or 15 mM.-   76. The formulation of any of aspects 71 to 75, wherein the    formulation comprises an excipient at a concentration of 1% to 20%.-   77. The formulation of aspect 76, wherein the excipient is a    disaccharide and/or a polyol.-   78. The formulation of aspect 76, wherein the excipient is a    non-reducing sugar.-   79. The formulation of aspect 77 or 78, wherein the excipient is    selected from mannitol, trehalose and sucrose.-   80. The formulation of any of aspects 76 to 79, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   81. The formulation of any of aspects 1 to 80, wherein the single    variable domain is stable under mechanical stress as determined by    visual inspection and/or OD320/OD280 measurement.-   82. The formulation of aspect 81, in which the OD320/OD280 is 0.05    or less.-   83. The formulation of any of aspects 81 or 82, wherein the    mechanical stress is selected from shaking during 10 s to 1 min,    pushing through a needle (25G, preferably 26G, more preferably 27G,    even more preferably 28G, most preferably 29G or more) with a    syringe, rotation for two days at 10 rpm, and stirring for 1 hour at    room temperature and/or 4-48 hours at 4° C. at at least 10 rpm (such    as 50 rpm, 100 rpm or more).-   84. The formulation of any of aspects 81 to 83, wherein the    formulation at least comprises one or more components selected from:-   b) An excipient at a concentration of 1% to 20%;-   c) A surfactant at a concentration of 0.001% to 1% selected from    TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.-   85. The formulation of any of aspects 81 to 84, wherein the    formulation comprises an excipient at a concentration of 1% to 20%.-   86. The formulation of aspect 85, wherein the excipient is a sugar    and/or polyol.-   87. The formulation of aspect 85, wherein the excipient is selected    from mannitol, glycine and sucrose, such as sucrose, or mannitol and    glycine.-   88. The formulation of any of aspects 85 to 87, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   89. The formulation of any of aspects 84 to 88, wherein the    formulation comprises a surfactant at a concentration of 0.001% to    1% selected from TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a    poloxamer.-   90. The formulation of aspect 89, wherein the formulation comprises    TWEEN (polysorbate) 80.-   91. The formulation of any of aspects 89 or 90, wherein the    surfactant has a concentration of 0.001% to about 0.1%, or about    0.01% to about 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%,    0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01%.-   92. The formulation of any of aspects 1 to 91, wherein the    concentration of polypeptide is about 1 to 200 mg/ml or more,    preferably about 5 to 100 mg/mL or more, more preferably about 5 to    50 mg/mL or more, most preferably about 5 to 30 mg/mL or more, such    as around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30    mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70    mg/mL, around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, around    150 mg/mL or even more.-   93. The formulation of any of aspects 1 to 92, wherein the aqueous    carrier is distilled water.-   94. The formulation of any of aspects 1 to 93, wherein the aqueous    carrier is MILLI-Q grade water or Water for Injection (WFI).-   95. The formulation according to any of aspects 1 to 94, which is    isotonic or slightly hypotonic.-   96. The formulation according to aspect 95, which has an osmolality    of 290±60 mOsm/kg.-   97. The formulation of any of aspects 1 to 96, wherein the    polypeptide comprises two or more single variable domains, such as    two or three.-   98. The formulation of any of aspects 1 to 97, wherein the    polypeptide specifically binds RANKL, IL-6R or IL-23.-   99. The formulation of aspect 98, wherein the polypeptide is    selected from one of SEQ ID NO's: 1 to 6.-   100. The formulation of any of aspects 1 to 99, wherein    -   the polypeptide has a solubility of at least 20 mg/mL,        preferably 50 mg/mL or more, more preferably 90 mg/mL or more or        120 mg/mL or more, most preferably 150 mg/mL or more, or even        200 mg/mL or more, as determined by the PEG exclusion method or        by a concentration experiment;    -   the polypeptide has a melting temperature of at least 59° C. or        more, preferably at least 60° C. or more, more preferably at        least 61° C. or more or at least 62° C. or more, most preferably        at least 63° C. or more as measured by the thermal shift assay        (TSA) and/or differential scanning calorimetry (DSC);    -   no particulates are present as measured by OD320/OD280 and/or        elastic light scattering; less than 10% of the polypeptide forms        pyroglutamate at the N-terminal glutamic acid during storage at        a temperature of 37±5° C. up to at least 2 weeks (preferably at        least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10        weeks, at least 3 months, at least 6 months, at least 1 year,        1.5 year or even 2 years or more), the % of pyroglutamate as        measured by RP-HPLC;    -   less than 10% of the polypeptide forms dimers during storage at        a temperature of 37±5° C. up to at least 2 weeks (preferably at        least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10        weeks, at least 3 months, at least 6 months, at least 1 year,        1.5 year or even 2 years or more), the % of dimers as measured        by SE-HPLC;    -   at least 80% of the polypeptide retains its binding activity to        at least one of its targets after storage at 37±5° C. up to at        least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at        least 2 months, at least 6 months, at least 1 year, 1.5 year or        even 2 years or more) compared to the binding activity prior to        storage, said binding activity as measured by ELISA and/or        BIACORE; and/or    -   the polypeptide is stable during mechanical stress.-   101. The formulation of any of aspects 1 to 100, wherein said    formulation comprises at least two components selected from:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%;-   c) A surfactant at a concentration of 0.001% to 1% selected from    TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.-   102. The formulation of any of aspects 1 to 101, wherein said    formulation comprises the following components:-   a) A buffer at a concentration of 10 mM to 100 mM selected from the    group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH    6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0;-   b) An excipient at a concentration of 1% to 20%;-   c) A surfactant at a concentration of 0.001% to 1% selected from    TWEEN (polysorbate) 80, TWEEN (polysorbate) 20 or a poloxamer.-   103. The formulation of any of aspects 1, 101 and/or 102, wherein    the pH is between 6.0 and 8, preferably between 6.2 and 7.5, more    preferably between 6.5 and 7.5 or 6.5 and 7.0, such as 6.5 or 7.0.-   104. The formulation of any of aspects 1, 101 to 103, wherein the    buffer is a histidine buffer.-   105. The formulation of aspect 104, wherein the buffer is a    histidine pH 6.5 or histidine pH 6.0 buffer.-   106. The formulation of any of aspects 104 or 105, wherein the    histidine buffer has a concentration of 10 to 50 mM, more preferably    10 to 20 mM.-   107. The formulation of any of aspects 104 to 106, wherein the    histidine buffer has a concentration of about 10 mM or about 15 mM.-   108. The formulation of any of aspects 1, 101 and/or 102 to 107,    wherein the excipient is a saccharide and/or polyol.-   109. The formulation of aspect 108, wherein the excipient is a    non-reducing sugar.-   110. The formulation of aspect 108, wherein the excipient is    selected from mannitol, trehalose, sorbitol and sucrose.-   111. The formulation of any of aspect 108 to 110, wherein the    excipient has a concentration of 2.5% to 15%, more preferably 5% to    10%.-   112. The formulation of aspect 111, wherein the excipient has a    concentration selected from about 5%, 7.5%, 8% and 10%.-   113. The formulation of any of aspects 1, 101 and/or 102 to 112,    wherein the surfactant is TWEEN (polysorbate) 80.-   114. The formulation of aspect 113, wherein the surfactant has a    concentration of 0.01% to 0.1%, preferably 0.01% to 0.05%.-   115. The formulation of aspect 114, wherein the surfactant has a    concentration of about 0.01% or 0.005%.-   116. The formulation of any of aspects 1 to 115, comprising:    a) A histidine pH 6.5 buffer at a concentration of 10 mM to 100 mM;    b) Sucrose at a concentration of 1% to 20%; and    c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.-   117. The formulation of aspect 116, comprising:    a) 15 mM histidine pH 6.5;    b) 8% sucrose; and    c) 0.01% TWEEN (polysorbate) 80.-   118. The formulation of aspect 117, comprising:    a) 15 mM histidine pH 6.5;    b) 8% sucrose;    c) 0.01% TWEEN (polysorbate) 80; and    d) A polypeptide selected from SEQ ID NO's: 1 to 6.-   119. The formulation of any of aspects 1 to 115, comprising:    a) A histidine pH 6.0 buffer at a concentration of 10 mM to 100 mM;    b) Sucrose at a concentration of 1% to 20%; and    c) TWEEN (polysorbate) 80 at a concentration of 0.001% to 1%.-   120. The formulation of aspect 119, comprising:    a) 10 mM histidine pH 6.0;    b) 10% sucrose; and    c) 0.005% TWEEN (polysorbate) 80.-   121. The formulation of aspect 120, comprising:    a) 10 mM histidine pH 6.0;    b) 10% sucrose;    c) 0.005% TWEEN (polysorbate) 80; and    d) A polypeptide selected from SEQ ID NO's: 1 to 6.-   122. A method for the preparation of a formulation of any of aspects    1 to 121, at least comprising the step of concentrating the    polypeptide and exchanging it with the selected buffer and/or    excipient.-   123. A sealed container containing a formulation according to any of    aspects 1 to 121.-   124. A pharmaceutical unit dosage form suitable for parenteral    administration to a human, comprising a formulation according to any    of aspects 1 to 121 in a suitable container.-   125. A kit comprising one or more of the sealed containers according    to aspect 123 and/or pharmaceutical unit dosage forms according to    aspect 124, and instructions for use of the formulation.-   126. The formulation, container, pharmaceutical unit dosage or kit    according to any of the preceding aspects for use in therapy.-   127. Method for prevention and/or treatment of one or more diseases    and/or disorders, comprising administering to a subject in need    thereof a formulation according to any of aspects 1 to 121.-   128. Method of aspect 127, wherein the disease is a disease and/or    disorder associated with aberrant expression and/or activity of    RANKL, disease and/or disorder associated with aberrant expression    and/or activity, such as overexpression of IL-6, or disease and    disorder associated with heterodimeric cytokines and their    receptors.-   129. Method of aspect 128, wherein the disease is selected from    osteoporosis, cancer induced bone loss and/or bone loss associated    with autoimmunity and/or viral infection.-   130. Method of aspect 128, wherein the disease is selected from    rheumatoid arthritis, abnormal synovial cell growth, plasmocytosis    induced Castleman's disease, tumor, muscle protein proteolysis,    multiple sclerosis, systemic lupus erythematosus, inflammatory bowel    disease, pancreatitis, psoriasis, angiogenesis, systemic-onset type    juvenile rheumatoid arthritis, spinal cord injury, endothelial    injury or destruction, mesothelioma, vasculitis, osteoarthritis,    inner ear disorder, cancer, rejection after transplantation,    pancreatic islet transplantation, myocardial infraction, prostate    cancer, choroidal neovascularization, muscle regeneration,    inflammatory myopathy, chronic rejection in cardiac transplant,    delayed graft function-   131. Method of aspect 128, wherein the disease is selected from    inflammation and inflammatory disorders such as bowel diseases    (colitis, Crohn's disease, IBD), infectious diseases, psoriasis,    cancer, autoimmune diseases (such as MS), carcoidis, transplant    rejection, cystic fibrosis, asthma, chronic obstructive pulmonary    disease, rheumatoid arthritis, viral infection, common variable    immunodeficiency.-   A-1. A stable NFD.-   A-2. A stable NFD in solution.-   A-3. A stable NFD obtainable by a process comprising the step of    concentrating a polypeptide comprising at least one single variable    domain and/or by a process comprising the step of storage of a    polypeptide comprising at least one single variable domain at    elevated temperature, e.g. at a temperature close to the melting    temperature or e.g. at 37° C. over a prolonged time period, e.g.    such as 1 to 4 weeks, e.g. 4 weeks.-   A-4. A stable NFD obtainable by a process comprising the step of    concentrating a polypeptide comprising and/or consisting of one or    more single variable domain(s) and one or more linkers.-   A-5. A stable NFD according to any of aspects A-3 or A-4, wherein    the step of concentrating is done by affinity- and/or ion exchange    chromatography.-   A-6. A stable NFD according to any of the aspects A-3 to A-5,    wherein the step of concentrating is done on a Protein A column, and    wherein high amounts of polypeptide are loaded on the column, e.g. 2    to 5 mg per ml resin Protein A.-   A-7. A stable NFD according to any of the aspects 5 or 6, wherein    the polypeptide is eluted by a steep pH gradient, e.g. a one step pH    change of 2.-   A-8. A stable NFD according to the previous aspects, wherein the NFD    is stable over a period of up to 2 years at −20 degrees Celcius.-   A-9. A stable NFD according to the aspects above, wherein the NFD is    stable over a period of up to 2 weeks at 4 degrees Celcius.-   A-10. A stable NFD according to the previous aspects, wherein the    NFD is stable over a period of up to 15 minutes at 50 degrees    Celcius.-   A-11. A stable NFD according to the previous aspects, wherein the    NFD is stable at acidic pH.-   A-12. A stable NFD according to the previous aspects, wherein the    NFD is stable at acidic pH over a prolonged period of time, e.g. a    time up to 1 day, more preferably 1 week, more preferably 2 weeks,    even more preferably 3 weeks, most preferred 4 weeks.-   A-13. A stable NFD according to the previous aspects, wherein the    NFD is stable at basic pH over a prolonged period of time, e.g. a    time up to 1 day, more preferably 1 week, more preferably 2 weeks,    even more preferably 3 weeks, most preferred 4 weeks.-   A-14. A stable NFD according to the previous aspects, wherein the    NFD is stable between pH 3 and pH 8.-   A-15. A stable NFD according to the previous aspects, wherein the    NFD is stable between pH 2.5 and pH 8.-   A-16. A stable NFD according to the previous aspects, wherein the    NFD is stable between pH 3 and pH 8 for 4 weeks at 4 degrees    Celcius.-   A-17. A stable NFD according to the previous aspects, wherein the    NFD is stable when mixing with organic solvents.-   A-18. A stable NFD according to the previous aspects, wherein the    NFD is stable when mixing with an alcohol, e.g. isopropanol.-   A-19. A stable NFD according to the previous aspects, wherein the    NFD is stable when mixing with 30% v/v of an alcohol, e.g.    isopropanol.-   A-20. A stable NFD according to the previous aspects, wherein the    dissociation constant of the binding of the NFD to its target    molecule is about the same as the dissociation constant of the    binding of its corresponding monomeric building block to said target    molecule.-   A-21. A stable NFD according to the previous aspects, wherein there    is no specific binding to its target molecule.-   A-22. A stable NFD according to the previous aspects, wherein the    dissociation constant of the binding of the NFD to its target    molecule is 30% or less, preferably 20% or less, more preferably 10%    or less, of the dissociation constant of the binding of its    corresponding monomeric building block to said target molecule.-   A-23. A stable NFD according to the previous aspects, wherein the    dissociation constant of the binding of the NFD to its target    molecule is 100 nM or less, preferably 10 nM or less, more    preferably 1 nM or less.-   A-24. A stable NFD according to the previous aspects, wherein the    koff value for the binding of the NFD to its target molecule is    about the same as the koff value for the binding of its    corresponding monomeric building block.-   A-25. A stable NFD according to the previous aspects, wherein the    koff value for the binding of the NFD to its target molecule is not    more than 90%, more preferably not more than 50%, even more    preferably not more than 40%, even more preferably not more than    30%, even more preferably not more than 20%, most preferably not    more than 10% higher than the koff value for the binding of its    corresponding monomeric building block.-   A-26. A stable NFD according to the previous aspects, wherein the    koff value for the binding of the NFD to its target molecule is not    more than 50% higher than the koff value for the binding of its    corresponding monomeric building block.-   A-27. A stable NFD according to the previous aspects, wherein the    koff value for the binding of the NFD to its target molecule is not    more than 10% higher than the koff value for the binding of its    corresponding monomeric building block.-   A-28. A stable NFD according to the previous aspects, wherein the    single variable domain is a Nanobody® such as a VHH, a humanized    VHH, an affinity-matured, stabilized, sequence optimized or    otherwise altered VHH or a construct thereof.-   A-29. A stable NFD according to the previous aspects, wherein the    single variable domain is selected from the group consisting of SEQ    ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,    SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID    NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, preferably SEQ ID NO: 8.-   A-30. A stable NFD according to the previous aspects, wherein the    single variable domain is selected from the group consisting of SEQ    ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,    SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID    NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, preferably SEQ ID NO: 8 and    of a functional sequence that is at least 70%, more preferably at    least 80%, even more preferably at least 90%, most preferably at    least 95% identical to any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:    9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ    ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID    NO: 20, preferably SEQ ID NO: 8-   A-31. A stable NFD according to the previous aspects, wherein the    single variable domain is selected from the group consisting of SEQ    ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,    SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID    NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, preferably SEQ ID NO: 8 and    of a functional sequence that is at least 70%, more preferably at    least 80%, even more preferably at least 90%, most preferably at    least 95% identical to any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:    9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ    ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID    NO: 20, preferably SEQ ID NO: 8 2; and wherein said polypeptide    specifically binds to its target molecule(s), more preferably has a    dissociation constant to at least one of its target molecules (if    bi- or multispecific), of 100 nM or less, even more preferably of 10    nM or less, most preferably of 1 nM or less.-   A-32. A NFD of any of the previous aspects (e.g. as described    herein) wherein the single variable domain is not VHH-R9 as    described in Spinelli et al, FEBS Letters 564 (2004) 35-40.-   A-33. A functional fragment of a NFD as described in any of aspects    A-1 to A-32.-   A-34. A polypeptide comprising at least one single variable domain,    wherein said at least one single variable domains can form a NFD as    e.g. described in any of aspects A-1 to A-32.-   B-1. A preparation comprising a NFD as described in any of aspects    A-1 to A-32, a functional fragment of aspect A-33, or a polypeptide    of aspect A-34.-   B-2. A preparation comprising a NFD as described in any of aspects    A-1 to A-32, a functional fragment of aspect A-33 or a polypeptide    of aspect A-34, wherein the ratio of NFD and its corresponding    monomeric building block is about 1 part NFD/1 part corresponding    monomeric building block to about 1 part NFD/2 parts corresponding    monomeric building block.-   B-3. A preparation comprising a NFD as described in any of aspects    A-1 to A-32, a functional fragment of aspect A-33 or a polypeptide    of aspect A-34, wherein the ratio of NFD and its corresponding    monomeric building block is about 1 part NFD/1 part corresponding    monomeric building block to about 2 parts NFD/1 part corresponding    monomeric building block.-   B-4. A preparation comprising a NFD as described in any of claims    A-1 to A-32, a functional fragment of aspect A-33 or a polypeptide    of aspect A-34, wherein the ratio of NFD and its corresponding    monomeric building block is 25% NFD/75% monomeric building block.-   B-5. A preparation comprising a NFD as described in aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, wherein the ratio of NFD and its corresponding    monomeric building block is 75% NFD/25% monomeric building block.-   C-1. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising a process step that has a condition that    favors hydrophobic interactions.-   C-2. A process of making a NFD according to aspect C-1, wherein said    process step is a purification step.-   C-3. A process of making a NFD according to aspect C-1, wherein    within said process step, the condition is such that it promotes    partial protein unfolding.-   C-4. A process of making a NFD according to aspect C-3, wherein said    process step is a purification step.-   C-5. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising the step of up-concentrating the monomeric    building blocks of said NFD, e.g. by binding the polypeptides    comprising one or more single variable domain(s) on an affinity    chromatography column, e.g. Protein A or IMAC.-   C-6. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising the step of binding polypeptides comprising    one or more single variable domain(s) on a affinity chromatography    column, e.g. Protein A or IMAC, and eluting with a pH step which    allows release of said polypeptide.-   C-7. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising the step of binding polypeptides comprising    one or more single variable domain(s) on a affinity chromatography    column, e.g. Protein A, and eluting with a pH step which allows    release of said polypeptide within 1 column volume.-   C-8. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising the step of ultra-filtration.-   C-9. A process according to aspect C-8, wherein the ultra-filtration    is done under conditions of low salt.-   C-10. A process of making a NFD according to any of aspects A-1 to    A-32, a functional fragment of aspect A-33 or a polypeptide of    aspect A-34, comprising the process step of storing the appropriate    polypeptide comprising one or more single variable domain(s) at    elevated temperature over a prolonged time.-   C-11. A process of making a NFD according to aspect C-10, wherein    said elevated temperature is 37° C. and time is 1, 2, 3, 4, 5, or 6,    preferably 4 weeks.-   C-12. A process of making a NFD according to any of aspect C-10 or    C-11, wherein said elevated temperature is such that it promotes    partial protein unfolding and exposure is over 1, 2, 3, 4, 5, or 6,    preferably 4 weeks.-   C-13. A process of making a NFD according to any of aspect C-10 to    C-12, wherein said elevated temperature is close to the melting    temperature of the polypeptide and exposure is over 1, 2, 3, 4, 5,    or 6, preferably 4 weeks.-   C-14. A process of making a NFD according to any of aspect C-9 to    C-13, wherein the CDR3 of said single variable domain is    destabilized.-   C-15. A process of making a NFD according to any of aspects C-10 to    C-14, wherein the single variable domain is a Nanobody®, such as    e.g. a VHH, a humanized VHH, an affinity-matured, stabilized,    sequence optimized or otherwise altered VHH.-   D-1. A process of making monomeric polypeptides comprising one or    more single variable domain(s), e.g. Nanobody® such as a VHH, a    humanized VHH, an affinity-matured, stabilized, sequence optimized    or otherwise altered VHH, wherein each of the steps in the making of    said polypeptide does not generate more than 10%, more preferably    not more than 5%, even more preferably not more than 4%, even more    preferably not more than 3%, even more preferably not more than 2%,    even more preferably not more than 1%, most preferred not more than    0.1% w/w corresponding NFD.-   D-2. A process according to aspect D-1, wherein each of the steps in    said process avoids conditions favoring hydrophobic interactions.-   D-3. A process according to any of aspects D-1 or D-2, wherein said    conditions favoring hydrophobic interactions is a high concentration    of the polypeptides, i.e. a concentration of the polypeptides e.g.    more than 10 mg polypeptide per ml resin column material; and thus a    process avoiding said interactions is avoiding such conditions in    each step of its making.-   D-4. A process according to aspect D-3, wherein column loads, e.g.    of an affinity column, are carefully evaluated and overload of the    column is avoided, i.e. a column load maximum should be determined    wherein not more than 10%, more preferably not more than 5%, even    more preferably not more than 4%, even more preferably not more than    3%, even more preferably not more than 2%, even more preferably not    more than 1%, most preferred not more than 0.1% w/w NFD is    generated.-   D-5. A process according to any of aspects D1 to D-4 of making    monomeric polypeptides comprising one or more single variable    domain(s), e.g. Nanobody® such as a VHH, a humanized VHH, an    affinity-matured, stabilized, sequence optimized or otherwise    altered VHH devoid of NFD or with no more than 50%, more preferably    no more than 40%, even more preferably no more than 30%, even more    preferably no more than 20%, most preferred no more than 10% NFD,    wherein each of the steps in said process avoids conditions favoring    hydrophobic interactions, e.g. wherein the process does not consist    of a protein A step and/or wherein said process avoids conditions    wherein the one or more single variable domain is partially    unfolded, e.g. CDR3 is destabilized and/or partially unfolded by    e.g. elevated temperature such as a temperature close to the melting    temperature of the polypeptide or e.g. 37° C., over a prolonged    time, e.g. weeks such as e.g. 4 weeks.-   E-1. A pharmaceutical formulation comprising a polypeptide    susceptible to dimerization (i.e. the formation of NFDs), e.g. a    polypeptide as described in any of aspects A-1 to A-31, e.g. a    polypeptide that comprises at least one of SEQ ID NO: 7, SEQ ID NO:    8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID    NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,    SEQ ID NO: 20, e.g. a polypeptide that comprises polypeptide B, and    polyol.-   E-2. The pharmaceutical formulation according to aspect E-1, wherein    the polyol is in a concentration of e.g. not more than 0.6M.-   E-3. The pharmaceutical formulation according to any of aspects E-1    or E-2, wherein the polyol is one or more selected from sorbitol,    mannitol, xylitol, ribitol, and erythritol.-   E-4. The pharmaceutical formulation according to any of aspects E-1    to E-3, wherein the polyol is mannitol, and e.g. in a concentration    of not more than 0.6 M mannitol.-   E-5. The pharmaceutical formulation according to any of aspects E-1    to E-4, wherein the polypeptide comprises a single variable domain    that binds serum albumin, preferably human serum albumin.-   E-6. The pharmaceutical formulation according to any of aspects E-1    to E-5, wherein the polypeptide comprises polypeptide B.-   E-7. The pharmaceutical formulation according to any of aspects E-1    to E-6, additionally comprising a non-reducing sugar such as e.g.    sucrose and/or trehalose and optionally NaCl and/or amino acids.-   E-8. The pharmaceutical formulation according to any of aspects E-1    to E-7, that is a liquid formulation.-   E-9. The pharmaceutical formulation according to any of aspects E-1    to E-8, that is prepared in a dried form, e.g. by lyophilization.-   E-10. The pharmaceutical formulation according to any of aspects E-1    to E-9, that is used as an injectable.-   E-11. The pharmaceutical formulation according to any of aspects E-1    to E-10, that is used as a subcutaneous formulation.-   F-1 A formulation, such as a pharmaceutical formulation, comprising    a polypeptide comprising one or more single variable domains, said    formulation being formulated for administration to a human subject,    further comprising an excipient at a concentration of 1% to 20%.-   F-2. A formulation comprising an aqueous carrier having a pH of 5.5    to 8.0 and a polypeptide comprising one or more single variable    domains at a concentration of 1 mg/mL to 200 mg/mL, said formulation    being formulated for administration to a human subject, and said    formulation further comprising an excipient at a concentration of 1%    to 20%.-   F-3. A formulation comprising an aqueous carrier having a pH of 5.5    to 8.0 and a polypeptide comprising one or more single variable    domains at a concentration of 1 mg/mL to 200 mg/mL, said formulation    being formulated for administration to a human subject, and said    formulation further comprising an excipient at a concentration of 1%    to 20%, wherein said formulation has an inorganic salt concentration    of 150 mM or lower.-   F-4. The formulation of any of aspect F-1 to F-3, wherein said    single variable domain is susceptible to dimerization.-   F-5. The formulation of aspect F-4, wherein the inorganic salt    concentration is from 50 mM to 100 mM or lower.-   F-6. The formulation of aspect F-5, that does not contain any    inorganic salt.-   F-7. The formulation of any of aspect F-1 to F-6, wherein the    polypeptide has a melting temperature of at least 59° C. or more    (such as 59.5° C. or more), preferably at least 60° C. or more (such    as 60.5° C. or more), more preferably at least 61° C. or more (such    as 61.5° C. or more) or at least 62° C. or more (such as 62.5° C. or    more), most preferably at least 63° C. or more (such as 63.5° C. or    more) as measured by the thermal shift assay (TSA) and/or    differential scanning calorimetry (DSC).-   F-8. The formulation of aspect F-7, wherein the formulation at least    comprises an excipient at a concentration of 1% to 20%.-   F-9. The formulation of aspect F-7, wherein the excipient is a    dissaccharide and/or a polyol.-   F-10. The formulation of aspect F-9, wherein the excipient is    selected from sucrose, mannitol, sorbitol and trehalose.-   F-11. The formulation of any of aspects F-8 to F-10, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   F-12. The formulation of any of aspects F-1 to F-11, wherein the    polypeptide is stable during storage at a temperature of 37±5° C. up    to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,    at least 8 weeks, at least 10 weeks, at least 3 months, at least 6    months, at least 1 year, 1.5 year or even 2 years or more), said    stability as determined by SE-HPLC.-   F-13. The formulation of aspect F-12, wherein less than 10%    (preferably less than 7.5%, more preferably less than 5%, most    preferably less than 2%) of the polypeptides forms dimers during    storage, the % of dimers as measured by SE-HPLC.-   F-14. The formulation of aspect F-13, wherein the formulation at    least comprises an excipient at a concentration of 1% to 20%.-   F-15. The formulation of aspect F-14, wherein the excipient is a    disaccharide and/or a polyol.-   F-16. The formulation of aspect F-14, wherein the excipient is a    non-reducing sugar.-   F-17. The formulation of aspect F-15 or F-16, wherein the excipient    is selected from trehalose, mannitol and sucrose.-   F-18. The formulation of any of aspects F-14 to F-17, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   F-19. The formulation of any of aspects F-12 to F-18, wherein at    least 80% (preferably at least 90%, more preferably at least 95% or    even at least 99%) of the polypeptides retain their binding activity    to at least one of their targets after storage compared to the    binding activity prior to storage, said binding activity as measured    by ELISA and/or BIACORE.-   F-20. The formulation of aspect F-19, wherein the formulation at    least comprises an excipient at a concentration of 1% to 20%.-   F-21. The formulation of aspect F-20, wherein the excipient is a    disaccharide and/or a polyol.-   F-22. The formulation of aspect F-20, wherein the excipient is a    non-reducing sugar.-   F-23. The formulation of aspect F-21 or F-22, wherein the excipient    is selected from mannitol, trehalose and sucrose.-   F-24. The formulation of any of aspects F-20 to F-23, wherein the    excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,    such as 5%, 7.5%, 8% or 10%.-   F-25. The formulation of any of aspects F-1 to F-24, wherein the    aqueous carrier is distilled water.-   F-26. The formulation of any of aspects F-1 to F-24, wherein the    aqueous carrier is MILLI-Q grade water or Water for Injection (WFI).-   F-27. The formulation according to any of aspects F-1 to F-26, which    is isotonic or slightly hypotonic.-   F-28. The formulation according to aspect F-27, which has an    osmolality of 290±60 mOsm/kg.-   F-29. The formulation of any of aspects F-1 to F-28, wherein the    polypeptide comprises two or more single variable domains, such as    two or three.-   F-30. The formulation of any of aspects F-1 to F-29, wherein the    polypeptide specifically binds serum albumin (preferably human serum    albumin), vWF, RANKL or IL-6R.-   F-31. The formulation of any of aspects F-1 to F-30, wherein the    polypeptide comprises at least a single variable domain that binds    serum albumin, preferably human serum albumin.-   F-32. The formulation of aspect F-31, wherein the polypeptide is    selected from one of SEQ ID NO's: 7 to 12 and 15 to 20.-   F-33. A method for the preparation of a formulation of any of    aspects F-1 to F-32, at least comprising the step of concentrating    the polypeptide and exchanging it with the selected buffer and    excipient.-   F-34. A sealed container containing a formulation according to any    of aspects F-1 to F-32.-   F-35. A pharmaceutical unit dosage form suitable for parenteral    administration to a human, comprising a formulation according to any    of aspects F-1 to F-32 in a suitable container.-   F-36. A kit comprising one or more of the sealed containers    according to aspect F-34 and/or pharmaceutical unit dosage forms    according to aspect F-35, and instructions for use of the    formulation.-   F-37. The formulation, container, pharmaceutical unit dosage or kit    according to any of the preceding aspects for use in therapy.-   F-38. Method for prevention and/or treatment of one or more diseases    and/or disorders, comprising administering to a subject in need    thereof a formulation according to any of aspects F-1 to F-32.-   F-39. Method of aspect F-38, wherein the disease and/or disorder is    a disease and/or disorder associated with aberrant expression and/or    activity of RANKL, disease and/or disorder associated with    overexpression of IL-6, or vascular disease and/or disorder.-   F-40. Method of aspect F-39, wherein the disease and/or disorder is    selected from osteoporosis, cancer induced bone loss and/or bone    loss associated with autoimmunity and/or viral infection.-   F41. Method of aspect F-39, wherein the disease and/or disorder is    selected from rheumatoid arthritis, abnormal synovial cell growth,    plasmocytosis induced Castleman's disease, tumor, muscle protein    proteolysis, multiple sclerosis, systemic lupus erythematosus,    inflammatory bowel disease, pancreatitis, psoriasis, angiogenesis,    systemic-onset type juvenile rheumatoid arthritis, spinal cord    injury, endothelial injury or destruction, mesothelioma, vasculitis,    osteoarthritis, inner ear disorder, cancer, rejection after    transplantation, pancreatic islet transplantation, myocardial    infarction, prostate cancer, choroidal neovascularization, muscle    regeneration, inflammatory myopathy, chronic rejection in cardiac    transplant, delayed graft function.-   F-42. Method of aspect F-39, wherein the disease and/or disorder is    selected from acute coronary syndrome (ACS), myocardial infarction,    thrombotic thrombocytopenic purpura (TTP) or Moschcowitz syndrome,    vascular surgery and stroke.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All of the references described herein are incorporated by reference, inparticular for the teaching that is referenced hereinabove.

EXAMPLES Example 1 Formulation and Stability Studies with RANKL008aExample 1.1 Materials and Methods Used in the Study 1.1.1 SingleVariable Domains

RANKL008a (SEQ ID NO: 4;EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS) has been described as SEQ ID NO: 759 inWO 2008/142164. RANKL008a is a trivalent bispecific Nanobody consistingof three humanized variable domains of a heavy-chain llama antibody, ofwhich two identical subunits are specific for binding to RANKL while theremaining subunit binds to HSA. The subunits are fused head-to-tail witha G/S linker in the following format: RANKL13H5-9GS-Alb8-9GS-RANKL13H5.

RANKL008a was expressed in Pichia pastoris and purified on SP SEPHAROSEas a capturing step and a Q filter as a polishing step or on SPSEPHAROSE as a capturing step and CAPTO MMC as a polishing step oralternatively by using a ProtA capture step followed by and SP SEPHAROSEpolishing step. Concentration of the Nanobody and buffer switch to PBS,10 mM phosphate+100 mM NaCl, 10 mM phosphate+5% mannitol or 10 mMphosphate+115 mM NaCl or others buffers was done via UF/DF or bydialysis. A final filtration on a 0.22 μm filter was performed. Thedifferent batches of RANKL008a ranged in concentration from 143 to 62.8mg/mL. Most batches were prepared at concentrations of about 60-85mg/mL.

1.1.2 Other Reagents

All reagents used in the study are standard chemical reagents of highestpurity. The complete composition of D-PBS was 137 mM NaCl, 2.7 mM KCl,10 mM Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic.

1.1.3 Equipment and Columns for Testing

HPLC experiments were carried out on an Agilent 1200 series instrumentfrom Agilent Technologies (Palo Alto, USA). The columns used were:

-   -   RP-HPLC: ZORBAX 300SB-C3 5-micron;    -   SE-HPLC: TSK-GEL G2000SW_(XL) (Tosoh Bioscience, Japan);    -   IEX-HPLC: Dionex PROPAC _(W)CX-10 column;

The concentration of the purified RANKL008a batches was determined byspectroscopy at 280 nm either using a NANODROP ND-1000(Thermoscientific), or a Uvikon 943 Spectrophotometer (KontronInstruments) or an Eppendorf Biophotometer 613.

Particle size distribution was measured on a PAMAS SVSS-C particlecounter (PArtikelMess-und AnalyseSysteme GMBH).

For potency measurements a BIACORE 3000 (GE Healthcare) was used. Elisabased potency assays were performed using standard laboratory equipmentand plate reader instrumentation.

Osmolality measurement was done with an osmometer Model 3320 fromAdvanced instruments.

1.1.4 Inhibition ELISA for RANKL Potency Measurement

RANKL008a interacts with human (soluble) receptor activator of nuclearfactor-kappaB ligand (RANKL) and blocks the interaction of this ligandwith its human receptor activator of nuclear factor-kappaB (RANK),thereby preventing signalization through this receptor. The potency ofRANKL008a was measured by an ELISA-based inhibition assay that allowsassessment of the relative potency of the RANKL binding moieties of anunknown batch of RANKL008a relative to that of a reference batch.

For the reference, the control and the test samples, different dilutionsof Nanobodies were prepared. These dilutions were pre-incubated with aconstant amount of 5 ng/mL soluble RANKL and a constant amount of 200ng/mL RANK-Fc. Subsequently, this mixture was transferred to amicrotiter plate coated with a non-blocking anti-Fc Nanobody. Afterwashing, residual bound RANKL was detected with a polyclonalbiotinylated anti-human RANKL antibody, followed by hors radishperoxidase (HRP)-labeled streptavidin detection.

The relative potency of the test samples compared to the referencesamples was analysed by use of PLA 2.0 Software (Stegmann Systems).

1.1.5 ELISA for HSA Binding

The relative potency of the HSA binding moiety in RANKL008a was measuredby an ELISA. Briefly, HSA was coated onto a plastic multiwell MAXISORPELISA plate by adsorption. After blocking excess binding sites on theplates with Superblock T20, a dilution series of references, control andtest samples was applied on the plate. Bound Nanobody was subsequentlydetected using a bivalent anti-Nanobody Nanobody, directly conjugated tohorseradish peroxidase (HRP).

The relative potency of the test samples compared to the referencesamples was analysed by use of PLA 2.0 Software (Stegmann Systems).

1.1.6 Purity Assay of RANKL008a by Size Exclusion High PerformanceLiquid Chromatography (SE-HPLC)

The SE-HPLC assay consisted of a pre-packed silica gel TSK-GELG2000SW_(XL) column, a mobile phase consisting of KCl, NaCl andphosphate buffer pH 7.2 (D-PBS) and UV detection at 280 nm. The relativeamount of the specific protein, variant, or impurities expressed as area%, was calculated by dividing the peak area corresponding to thespecific protein or to any protein impurity by the total area of allintegrated peaks.

1.1.7 Purity Assay of RANKL008a by Reverses Phase High PerformanceLiquid Chromatography (RP-HPLC)

In the RP-HPLC assay a ZORBAX 300SB-C3 column (Agilent Technologies,Palo Alto, US) was used. The relative amount of a specific proteinimpurity was determined by measuring the light absorbance of thecomponents eluting from the RP-HPLC column. The relative amount of thespecific protein, variant, or impurities expressed as area %, wascalculated by dividing the peak area corresponding to the specificprotein or to any protein impurity by the total area of all integratedpeaks.

1.1.8 Purity Assay of RANKL008a by Ion Exchange High Performance LiquidChromatography (IEX-HPLC)

The IEX-HPLC assay combined the use of a pre-packed Dionex PROPAC WCX-10weak cation exchange column, a mobile phase consisting of citrate bufferpH 5.5 and UV detection at 280 nm. After loading the protein(s) on thecolumn, bound materials were eluted by a sodium chloride gradient. Therelative amount of the specific protein, variant, or impuritiesexpressed as area %, was calculated by dividing the peak areacorresponding to the specific protein or to any protein impurity by thetotal area of all integrated peaks.

1.1.9 Relative Potency Determination on BIACORE

RANKL or HSA was immobilized on the BIACORE chip (amine coupling usingthe BIACORE amine coupling kit). After a preconditioning step of 5injections of RANKL008a, all samples were diluted to 2.5 nM intriplicate and analyzed on the chip. Slopes were determined using thegeneral fit method and the linear fit model (BIAevaluation software). Todetermine the initial binding rate (IBR), the slope between 5 s and 30 swas selected. The values of these slopes were transferred in excel andthe percentage activity/potency compared to the RANKL008a referencematerial was determined. BIACORE potency is thus expressed as relativepotency compared to the reference materials.

Example 1.2 Stability of the Nanobody in Different Buffers afterDifferent Freeze/Thaw Cycles

A freeze/thaw stability study was performed to determine the effect ofrepetitive freeze and thawing on the recovery, physical stability andchemical stability of RANKL008a. Aliquots of batch RANKL008a formulatedat ˜60-85 mg/mL in the buffers 1-12 given in Table 1 were subjected to10 freeze/thaw (F/T) cycles at −20° C. One F/T cycle is defined byfreezing the sample for 1 hour in a freezer at −20° C. followed bythawing at room temperature for 30 minutes. The stressed samples werecompared with reference material (stored at 4° C.) using SE-HPLC (FIG. 1(A); representative figure of the experiments performed in phosphatebuffer), RP-HPLC (FIG. 2; representative figure of the experimentsperformed in phosphate buffer) and the ELISA potency assays (Table 2).All other data of the freeze thaw experiments demonstrate similarpatterns as given in FIGS. 1 and 2 (except FIG. 1 (B) see below).

Subjecting RANKL008a to 10 F/T cycles had no significant effect on itsstability: the SE-HPLC- and RP-HPLC profiles were comparable between thereference batches and material subjected to multiple freeze/thaw cycles.There was no decrease in the total surface area and no new peaks werebeing formed, except in 20 mM L-histidine, pH 5.5+10% mannitol and 20 mML-histidine, pH 6+10% mannitol, where the main peak had a very smallshoulder in the SE-HPLC chromatograms (FIG. 1 (B) showing the data at pH5.5 and where the minor shoulder on the main peak is indicated by anarrow). In Table 3 and Table 4 data are included for the integration ofthe different peaks (expressed as % surface area) in the SE-HPLC andRP-HPLC analysis respectively. These data demonstrate that no changesoccur in the profiles after 10 freeze thaw cycles.

Analysis by the ELISA potency assays indicated no loss of activity afterrepetitive freezing and thawing in all formulations, except in 20 mMHistidine, pH 5.5+10% mannitol where there appeared to be a lower RANKLand HSA binding potency.

Example 1.3 Stability of the Nanobody in Different Buffers when Storedat 37° C. Up to 10 Weeks

RANKL008a was formulated in different buffers at 60-85 mg/mL (buffers1-12 given in Table 1). The stability of the different samples wasassessed in accelerated stress conditions at 37° C.±3° C. Samples weretaken after 2, 3, 5 and 10 weeks storage at this temperature and wereanalyzed using SE-HPLC, RP-HPLC and IEX-HPLC. BIACORE was performed onthe samples stored for 10 weeks to evaluate loss in potency.

1.3.1 SE-HPLC Analysis

The results of the analysis of a sample by SE-HPLC is given in FIG. 3where an example is shown for the sample stored during two weeks at 37°C. in the presence of 50 or 100 mM salt or 10% mannitol phosphatebuffer. Storage at 37° C. resulted in the formation of a clear prepeakeluting at about 40 minutes and some minor postpeaks close to the mainpeak; at the 60 minutes elution time (see insert in FIG. 3) somedegradation fragments could be observed. In Table 3 the integration datafor all samples analysed is summarized for the different peaks observed(except peaks after 60 minutes elution time). The peak area of theprepeak increased over time but was reduced by the addition of mannitolto the buffer (Table 3). The postpeaks after 60 minutes elution timecorresponded to degradation products (due to remaining proteolyticactivity in sample). The relative area (%) of these peaks increased onlyslightly, implying that degradation was restricted to a minimum.

The prepeak represented the dimeric form of RANKL008a. The peak surfacearea of the prepeak increased with storage time (Table 3) and wasaccompanied by a concomitant decrease in surface area of the main peak(Table 3). The propensity to form dimers was significantly lower in theformulations containing 10% mannitol, which seemed to have a positiveeffect in suppressing the dimerization process. Note the significantlower amounts of dimers observed in the Acetate and Histidine buffers(pH 5.5) containing 10% mannitol (Table 1 and FIG. 4). FIG. 4(A)summarizes the % surface area for the main peak in the different buffersand at different time points when stored at 37° C. FIG. 4(B) summarizesthe data for the % prepeak (dimer).

1.3.2 RP-HPLC Analysis

A representative RP-HPLC is given in FIG. 5 where the RP-HPLCchromatogram of a RANKL008a sample stored in phosphate buffer withdifferent concentrations salt or 10% mannitol is represented. TheRP-HPLC profiles of RANKL008a formulated in the 12 different bufferswere comparable to this Figure. In Table 4 integration data for thedifferent peaks detected is summarized. In FIG. 5 the inset shows a zoomon the main peak where the two pre-peaks can be discriminated, while thepost peak that is fully base line resolved from the main peak is thepyro-glutamate variant of the RANKL008a where the N-terminal glutamicacid has been converted to the pyroglutamate form.

There were two differences between the RP-HPLC profiles of the referencebatch and the storage samples. Firstly, the pyroglutamate peak increasedwith increased incubation time and was more apparent in phosphate bufferat pH 7 than at pH 5.5 or pH 6. Secondly, a prepeak was being formed infunction of storage time. The surface area of this peak was higher inthe phosphate buffer.

There were no differences in the RP-HPLC profiles of the samples withoutor with mannitol.

1.3.3 IEX-HPLC Analysis

A representative IEX-HPLC chromatogram of the RANKL008a stored for 2weeks in phosphate buffer with different salt concentrations or 10%mannitol is depicted in FIG. 6. The inset shows a zoom in on the mainpeak where a minor post peak 1 and a more significant post-peak 2 isobserved. Results of the analysis of the different samples by IEX-HPLCare given in Table 5 and FIG. 7.

The first postpeak constituted maximally 4.5% of the total peak surfacearea. The surface area of this peak was the highest in phosphate bufferand the lowest or even absent in the mannitol-containing buffers. Thepeak area of the second peak on the other hand was substantial, yetsignificantly lower in the buffers containing 10% mannitol (FIG. 7). Thematerial eluting in the post-peak was collected by fraction collectionand re-chromatographed on the SE-HPLC column described above. Thispost-peak 2 elutes in the SE-HPLC chromatogram at the dimer positiondemonstrating that i) this dimer does not dissociate under theseconditions and that ii) this dimer elutes later on the IEX-HPLC column.Therefore we can conclude that the post-peak 2 is the dimerized form ofthe RANKL008a.

1.3.4 BIACORE Potency Analysis of the RANKL008a Stored at 37° C.

The RANKL and HSA binding of RANKL008a in stability samples stored for10 weeks at 37° C. was compared with the activity of the unstressedreference batch using BIACORE analysis. The relative potencies are givenin Table 6 and are expressed as % activity compared to reference batch.

After 10 weeks of storage at 37° C. the relative potency of RANKL008afor binding RANKL had dropped to 70-80% in the different buffers (Table6). In histidine, pH 6+10% mannitol, the activity remained the highest(87.4%). The higher the NaCl concentration in the buffer, the lower therelative potency in the sample (compare the values obtained in bufferswith 50 mM NaCl and 100 mM NaCl in Table 6).

The relative potency for HSA binding had dropped more compared to theactivity for RANKL binding after 10 weeks storage at 37° C. Thisdecrease in activity however was less significant in themannitol-containing buffers than in the NaCl-containing buffers. Asobserved for RANKL binding, the percentage activity on HSA decreasedwith increasing concentrations of NaCl in the different buffers.

Example 1.4 Osmolality Measurement for the Nanobodies in the DifferentBuffers

Osmolality measurements were performed on the different formulationsused in the stability studies:

-   -   RANKL008a in 10 mM Phosphate/10% mannitol: 635 mOsm/kg    -   RANKL008a in 10 mM Acetate/10% mannitol: 643 mOsm/kg    -   RANKL008a in 20 mM L-histidine pH 5.5/10% mannitol: 712 mOsm/kg    -   RANKL008a in 20 mM L-histidine pH 6.0/10% mannitol: 667 mOsm/kg    -   RANKL008a in 10 mM acetate buffer pH 5.5/100 mM NaCl: 272        mOsm/kg    -   RANKL008a in 10 mM phosphate/5% mannitol: 389 mOsm/kg

Formulations containing 10% mannitol were hypertonic.

Example 1.5 Stability of the Nanobodies During Mechanical Stress

Mechanical stress experiments were performed on RANKL008a (62.2 mg/mL)in 10 mM phosphate buffer pH 7.0 with 115 mM NaCl. The RANKL008a samplewas diluted (in the 10 mM phosphate buffer pH 7.0 with 115 mM) orundiluted with and without 0.01% TWEEN (polysorbate) 80. The sampleswere shaken, stirred, rotated and pushed through a needle with a syringe(the syringe used can be any commercially available syringe, such ase.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL up to 50mL syringe) as follows:

-   -   Diluted to 5 mg/mL or undiluted and shaken (10 s-1 min);    -   Pushed through a syringe (3 mL) with needle 25G (undiluted)        (10×);    -   Rotated (10 rpm) on an end over end mixer for 2 days at room        temperature (undiluted)    -   Stirred 1 hour at room temperature for 2 days at 5° C. (diluted        to 5 mg/mL)        The different samples were compared visually for any differences        in appearance.

Strong shaking for a short time (10 s) caused strong foaming of thesamples in the absence of TWEEN (polysorbate) 80, the diluted sample gotvery opaque (FIG. 8) while this was less pronounced for the undilutedsample. In the presence of the TWEEN (polysorbate) 80 this opacity wasnot observed.

The undiluted RANKL008a sample with and without 0.01% TWEEN(polysorbate) 80 was pushed 10 times through a needle (25G) with a 3 mLsyringe. The sample without TWEEN (polysorbate) 80 got opaque, there wasalso formation of foam and tiny air bubbles were visible when tappingthe vial, in the vial with 0.01% TWEEN (polysorbate) 80 opacity waslimited.

The undiluted RANKL008a sample with or without 0.01% TWEEN (polysorbate)80 was rotated for 2 days at 10 rpm. Both samples stayed clear.

The diluted (to 5 mg/mL) RANKL008a sample with or without 0.01% TWEEN(polysorbate) 80 was stirred for 1 hour at room temperature and furtherfor 2 days at 5° C. The visual observations are as follows: stirringduring 1 h at room temperature induced an opacity which was not observedin the presence of 0.01% TWEEN (polysorbate) 80. After stirring 1 hourat room temperature, the sample without TWEEN (polysorbate) 80 wasslightly opaque while the sample with TWEEN (polysorbate) 80 stayedclear. After 2 days stirring at 5° C., both sample were opaque but theopacity in the sample without TWEEN (polysorbate) 80 was higher.

With the addition of 0.01% TWEEN (polysorbate) 80, the RANKL008a samplewas less or not opaque after mechanical stress and there was less foamformation. This indicates that the sample is less susceptible todenaturation at the air-water interface if TWEEN (polysorbate) 80 isadded.

Example 1.6 Syringeability of the Different Nanobody Formulations

The effect of using different diluents—i.e. saline solution, phosphatebuffer without TWEEN (polysorbate) 80 or phosphate buffer with TWEEN(polysorbate) 80—on content, visual appearance and potency of RANKL008aat low concentration (0.28 mg/mL) was determined after passage throughdifferent syringes and needles. RANKL008a was diluted in differentdiluents followed by passage or 24 h storage in syringes (BectonDickinson) (FIG. 9). FIG. 9 contains the legends to the differentsamples generated during this experiment where the following codes areapplied:

S25/0: storage at 25° C. for 0 minute

525/24: storage at 25° C. for 24 h

−TW: buffer minus TWEEN (polysorbate) 80

+TW: buffer+TWEEN (polysorbate) 80

PLACEBO refers to the following buffer: 10 mM Na₂HPO₄ pH 7.0+115 mM NaCl

TUB: sample stored in a polystyrene tube

Visual inspection and content determination of RANKL008a after dilutionin different diluents and passage/storage in syringes is given in Table7. Data on turbidity measurement at 320 and 350 nm are given in FIG. 10.The relative HSA and RANKL potency of RANKL008a after dilution indifferent diluents and passage/storage in syringes is shown in FIG. 11.

Passage through a syringe slightly increased turbidity when using 10 mMNa₂HPO₄, 115 mM NaCl (pH 7.0). Dilution in saline solution caused a dropin RANKL/HSA binding activity of 18-34.0%. A similar effect was observedusing 10 mM Na₂HPO₄, 115 mM NaCl (pH 7.0) without TWEEN (polysorbate)80, i.e. a drop of 15-27%. In contrast, dilution in 10 mM Na₂HPO₄, 115mM NaCl (pH 7.0) with TWEEN (polysorbate) 80 did not appear to have adramatic effect confirming the beneficial role of TWEEN (polysorbate) 80in the buffer.

Example 1.7 Stability of Nanobody Formulations During Syringe Passagewith Different Needle Size

The effect of syringe passage on visual appearance of the RANKL008ausing different needle sizes and needle size combinations was evaluated.RANKL008a was diluted in an Eppendorf tube (TUB) in 10 mM Na₂HPO₄, 115mM NaCl, 0.01% TWEEN (polysorbate) 80 (v:v), (pH 7.0) to a finalconcentration of 0.28 mg/mL followed by single passage through a 1 mLBecton Dickinson syringe 5 equipped with different needles (i.e. Terumo18G6, 23G, 27G and 30G) (FIG. 12). In this Figure and Table 8 thefollowing codes apply:

+TW: buffer+TWEEN (polysorbate) 80

PLACEBO refers to the following buffer: 10 mM Na₂HPO₄ pH 7.0+115 mM NaCl

TUB: sample stored in a polystyrene tube

18G/18G: sample drawn up with a 18G needle and expelled through a 18Gneedle

18G/27G: drawn up with a 18G needle and expelled through a 27G needle

All other coding is similar to the two examples given above.

Turbidity was determined by visual inspection and by measurement of theabsorption at wavelengths of 320 nm, 340 nm, 350 nm and/or 500 nm, anddetermining the ratio of the obtained value over the absorption at A278nm (mostly 320/278 and 350/278). A ratio of >0.05 was consideredsignificant. Visual inspection, content and turbidity of RANKL008abefore (TUB) and after passage through syringes with different needlesize is shown in Table 8.

In a further experiment, RANKL008a was subjected to single passagethrough a 1 mL syringe equipped with different needle sizes (i.e. 27Gand 29G) both undiluted (65 mg/mL) and diluted in 10 mM Na₂HPO₄, 115 mMNaCl, 0.01% TWEEN (polysorbate) 80 (v:v), (pH 7.0) to a finalconcentration of 0.28 mg/mL (FIG. 13). Visual inspection, content andturbidity of RANKL008a before (TUB) and after passage through syringeswith different needle size is shown in Table 9.

In Table 9 and FIG. 13 the following codes are used:

-   -   +TW: buffer+TWEEN (polysorbate) 80    -   PLACEBO refers to the following buffer: 10 mM Na₂HPO₄ pH 7.0+115        mM NaCl    -   TUB: sample stored in a polystyrene tube    -   27G/27G: sample drawn up with a 27G needle and expelled through        a 27G needle    -   29G/29G: drawn up with a 29G needle and expelled through a 29G        needle    -   T: Terumo needle, B Becton Dickinson needle    -   0028 refers to concentration at 0.28 mg/mL, 6500 to about 65        mg/mL

Different combinations of needle sizes did not have a significant effecton RANKL008a recovery or sample turbidity both at low (0.28 mg/mL) andhigh (62.8 mg/mL) concentration (up to gauge sizes 29 and 27respectively). In both experiments turbidity values were low (<0.05).

Example 2 Formulation and Stability Studies with Nanobodies that BindIL-6R Example 2.1 Materials and Methods Used in the Study 2.1.1 SingleVariable Domains

Three Nanobodies that were used in this study have been described in PCTapplication No. PCT/EP2010/054764 to Ablynx N.V. IL6R304 is a bispecificNanobody consisting of two humanized variable domains of a heavy-chainllama antibody, one binding to IL-6R, the other one (Alb8) binding toHSA. The trivalent bispecific Nanobodies IL6R305 and IL6R306 consist oftwo identical subunits that are specific for IL-6R while the thirdsubunit binds to HSA. The build-up of the subunits differs in IL6R305and IL6R306 (see Table C-27 of PCT/EP2010/054764). The subunits in allthree Nanobodies are fused head to-tail with a 9G/S linker. Thesequences and characteristics of the three Nanobodies are given in Table10.

The Nanobodies were expressed in Pichia pastoris. Concentration of theNanobody and buffer switch to PBS or other formulation buffer was donevia UF/DF (Sartorius Hydrosart SARTOCON Slice 200, 10 kDa). A finalfiltration was carried out at 0.22 μm. An overview of the differentIL-6R Nanobody batches is given in Table 11.

Unstressed samples in PBS or other formulations were used as referencematerial for analyzing the storage stability samples.

2.1.2 Other Reagents

Reagents used in the study are given in Table 12. The completecomposition of D-PBS was 137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphatedibasic, 2 mM Potassium Phosphate monobasic.

2.1.3 Equipment and Methods for Measurements

HPLC experiments were carried out on an Agilent 1200 series instrumentfrom Agilent Technologies (Palo Alto, USA) or on a Dionex Ultimate 3000instrument. The columns used were:

-   -   RP-HPLC: ZORBAX 300SB-C3 5-micron, 4.6×150 mm (Agilent, Cat. No.        883995-909) or ZORBAX 300SB-C8 5-micron, 4.6×150 mm (Agilent,        Cat. No. 883995-906);    -   SE-HPLC: Phenomenex BIOSEP SEC S2000 (OOH-2145-KD)

Concentration determinations of the Nanobodies were done with NANODROPND-1000 (Thermoscientific), with a Uvikon 943 Spectrophotometer (KontronInstruments) or with an Eppendorf Biophotometer 6131 at 280 nm.

Particle size distribution was measured on a PAMAS SVSS-C particlecounter (PArtikelMess-und AnalyseSysteme GMBH).

Osmolality measurement was done with an osmometer Model 3320 fromAdvanced instruments.

For measurement of binding activity BIACORE 3000 (GE Healthcare) wasused.

The thermal shift assay (TSA) was performed on a UghtCycler480 Q-PCRdevice (Roche).

For determination of the Tm, an automated VP-capillary DifferentialScanning Calorimeter (DSC, MicroCal) was used.

2.1.4 Purity Assay of the IL-6R Nanobodies by Size Exclusion HighPerformance Liquid Chromatography (SE-HPLC)

The SE-HPLC assay consisted of a pre-packed Phenomenex BIOSEP SEC S2000column, a mobile phase consisting of KCl, NaCl and phosphate buffer pH7.2 (D-PBS) and UV detection at 280 nm. The relative amount of specificprotein impurity was expressed as area %, and was calculated by dividingthe peak area corresponding to the protein impurity by the totalintegrated area.

The method can resolve and quantify the relative amounts of intactmaterial and product related impurities such as aggregates anddegradation fragments.

2.1.5 Purity Assay of the IL-6R Nanobodies by Reverses Phase HighPerformance Liquid Chromatography (RP-HPLC)

In the RP-HPLC assay a ZORBAX 300SB-C3 or ZORBAX 300SB-C8 column(Agilent Technologies, Palo Alto, US) at elevated temperatures wereused. With the C3 column, mobile phase A consisted of 0.1% TFA andmobile phase B consisted of 0.1% TFA in ACN/isopropanol. With the C8column, mobile phase A consisted of 0.1% TFA and mobile phase Bconsisted of 0.1% TFA in 1-propanol. The relative amount of a specificprotein impurity was determined by measuring the light absorbance (280nm) of the components eluting from the RP-HPLC column. The relativeamount of a specific protein impurity, expressed as area %, wascalculated by dividing the peak area corresponding to the impurity bythe total integrated area.

2.1.6 Measurement of Particle Size Distribution (PAMAS)

The measurements on the PAMAS SVSS-C particle counter were performed asfollows: 100 μl sample was diluted 1/10 in 1 mL MILLI-Q water and 10consecutive measurements were performed in all 16 channels (diameter set1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100, 150 and 200 μm). Forcalculation of the average value, the first 2 measurements were excludedand the dilution factor was taken into account. The results are given ascumulative data (total particle counts>x μm) or differential data (totalparticle counts between diameter x and y μm). Only the cumulative dataare presented.

2.1.7 Affinity Measurement on BIACORE

A chip was first immobilized with HSA (amine coupling using the BIACOREamine coupling kit). After a preconditioning step of 5 injections of theNanobody, all samples were diluted to 2.5 nM in triplicate and analyzedon the chip. Quality control of the chips using the reference sample wasincluded in the experiment to detect any loss of activity or decrease inresponse (deterioration of the chip). Slopes were determined using thegeneral fit method and the linear fit model (BIAevaluation software). Todetermine the initial binding rate (IBR), the slope between 5 s and 30 swas selected. The values of these slopes were transferred in excel andthe percentage activity compared to the reference was determined.

2.1.8 ELISA Based Potency Assay for HSA Binding

Human Serum Albumin (HSA) was immobilized onto a multiwell MAXISORPELISA plate by adsorption. After blocking excess binding sites on theplates with Superblock T20 (PBS) blocking buffer, a dilution series oftest and reference samples was applied on the plate. Bound Nanobody wassubsequently detected using a bivalent anti-Nanobody Nanobody directlyconjugated to horseradish peroxidase (HRP). In the presence of H₂O₂ HRPcatalyzes a chemical reaction with Tetramethylbenzidine (es TMB) whichresults in the formation of a color. The reaction was stopped by adding1N HCl. The optical density of the color was measured at 450 nm.

2.1.9 ELIAS Based Potency Assay for IL-6R Binding

For the reference, control and test samples, different dilutions of theNanobodies were prepared. These dilutions were pre-incubated with aconstant amount of 100 ng/mL IL-6, followed by the addition of 4 ng/mLsoluble IL-6R. Subsequently, this mixture was transferred to amicrotiter plate coated with a non neutralizing anti-IL-6R Nanobody.After washing, residual bound IL-6 was detected with biotinylatedanti-human IL-6 monoclonal antibody, followed by HRP-labeledstreptavidin detection. In the presence of H₂O₂ HRP catalyzes a chemicalreaction with Tetramethylbenzidine (es TMB) which results in theformation of a color. The reaction was stopped by adding 1N HCl. Theoptical density of the color was measured at 450 nm. The relativepotency of the test samples compared to the reference sample wasanalyzed by use of PLA 2.0 Software.

2.1.10 Capillary Isoelectric Focusing (cIEF)

Capillary Isoelectric Focusing (cIEF) is an analysis/separationtechnique that differentiates proteins with respect to charge, i.e., itseparates proteins according to their isoelectric points (pI). Theseparation principle is similar to gel-based/flatbed IEF but differsmainly in its format, that is, the separation takes place in an opentube of narrow format (capillary) eliminating the need for anyanticonvective matrix support. Also, cIEF is a fully automatedinstrument with online detection and data acquisition. A drawback oftraditional cIEF in a conventional CE instrument is that the focused(stationary) zones must be mobilized past the single-point detectionarea in order to record the signal. During mobilization the zones maybecome broadened with contaminant loss of resolution and decreaseddetectability. Moreover, the analysis time and risk of proteinaggregation/precipitation will increase. By imaging cIEF the focusingprocess is followed in real-time over the whole-column/capillary by aCCD camera excluding the mobilization step. As soon as the focusingprocess is completed the analysis run is finished.

Example 2.2 Tm Determination

The melting temperature (Tm) in different buffers was determined usingthe fluorescence-based thermal shift assay (TSA, for IL6R304 andIL6R305) and by differential scanning calorimetry (DSC, for IL6R304).

2.2.1 Thermal Shift Assay

The thermal shift assay or TSA can be performed in 96-well plate in aQ-PCR device to evaluate the effect of buffer couple, ionic strength, pHand excipients on the thermal stability of proteins. The assay resultsin a Tm value that is indicative for the thermal stability in the testedbuffers. Briefly, the assay follows the signal changes of a fluorescencedye, such as SYPRO Orange, while the protein undergoes thermalunfolding. When SYPRO Orange is added to a properly folded proteinsolution, it is exposed in an aqueous environment and its fluorescencesignal is quenched. When the temperature rises, the protein undergoesthermal unfolding and exposes its hydrophobic core region. SYPRO Orangethen binds to the hydrophobic regions, unquenches which results in theincrease of the fluorescence signal.

In a first experiment, the Tm was assessed for IL6R304 and IL6R305 indifferent buffers, excipients and combinations thereof using the TSAassay. The obtained Tm values are displayed graphically in FIG. 14 andFIG. 15.

In all conditions tested, the Tm values were slightly higher for IL6R304than for IL6R305. The buffers and excipients tested had a similar effecton the Tm values of IL6R304 and IL6R305:

Effect of buffer pH: the highest melting points were obtained in Hepesbuffer (pH 7 and pH 8) and L-histidine pH 6.5, followed by phosphatebuffer (pH 6.7 and 7.7), Tris pH 7.2 and succinate buffer pH 6.2. Thelowest melting points were obtained in PBS (58.82° C. for IL6R304) andin the buffers with the lowest pH, i.e. succinate pH 5.2 and L-histidinepH 5.5. The higher melting point in L-histidine pH 6.5 correlates wellwith the higher solubility of IL6R304 in this buffer (see Example 2.3).

Effect of [NaCl] concentration: the highest melting temperatures weremeasured when no sodium chloride was added to the buffers. Tm valuesdecreased gradually with increasing NaCl concentration; the effectplateaus at 300 mM NaCl.

Effect of the excipients mannitol, sucrose and glycine: all excipientstested appeared to have a stabilizing effect on IL6R304 and IL6R305,since the melting temperatures increased with increasing excipientconcentration. The highest Tm values were obtained in buffers containing7.5% mannitol or 5% sucrose.

In summary, IL6R304 and IL6R305 seemed to be more stable in buffers thathad a neutral pH and which contained a low NaCl concentration andsignificant amounts of mannitol or sucrose. This is representedschematically in Table 13. L-histidine pH 6.5 appeared to be a goodbuffer to take forward in further stability testing and formulationwork. Another important argument for proceeding with a L-histidinebuffer is that the solubility of IL6R304 was shown to increasedramatically in this buffer compared to PBS (see Example 2.3). Bufferscontaining high NaCl concentration should be avoided, while Hepesbuffers at neutral pH could preferably be used.

2.2.2 Differential Scanning Calorimetry

To identify other suitable buffers to be used in purification protocols,i.e. in which the protein displays acceptable stability, differentialscanning calorimetry (DSC) was used to determine the melting temperatureof IL6R304 in different candidate buffers.

FIG. 16 shows the results of a DSC experiment performed on IL6R304formulated in different buffers. The highest melting temperatures wereobserved in MES (pH 6.0) and Hepes (pH 7.0), whereas acetate, Tris-HCland phosphate indicate a slightly lower thermal stability. The Tm valueobtained in citrate (pH 3.5) was on average 10° C. lower than in theother buffers. All heat capacity (Cp) melting peaks were sharp andrather symmetrical in all buffers. The restoration of a base-line afterthe transition indicated that no precipitation had occurred.

An overview of all Tm values obtained in the DSC experiments is shown inTable 14. From these results we can conclude that adding more NaCl leadto a gradual decrease in melting temperature, as was also observed inthe TSA.

2.2.3 TSA Experiment Using Experimental Design

A second TSA experiment was performed on IL6R304 using experimentaldesign (DOE). The DOE consisted of different steps. The outcome of eachstep was used to define the DOE for the next step. Briefly, theexperimental set-ups and obtained results were the following:

-   -   Step 1: L-histidine, succinate, phosphate and Tris were tested        at different pH (6-7) and buffer strength (10-40 mM). The most        promising formulation was found to be low ionic strength        L-histidine and Phosphate buffers with a pH of 7 and 6,        respectively.    -   Step 2: the effect of adding NaCl and different excipients to 15        mM L-histidine pH 6.5 and 7 and 15 mM Phosphate pH 6 and 6.5 was        tested. One representative of three excipient families was        included: mannitol (polyol), sucrose (non-reducing sugar) and        arginine (amino acid). It was concluded that Arginine and/or        NaCl decrease the Tm of IL6R304. A maximal melting temperature        was reached in sucrose or a mix of sucrose/mannitol (10% in        total). Furthermore, a higher Tm was obtained in L-histidine        than in phosphate. For both buffers, best results were obtained        at pH 6.5.    -   Step 3: other representatives, i.e. sorbitol, xylitol, ribitol,        trehalose and glycine, from the three excipient families were        tested in 15 mM L-histidine and 15 mM phosphate, both at pH 6.5.        Again, the Tm of IL6R304 was higher in L-histidine buffer than        in phosphate for all excipients and excipient combinations. The        best buffer formulation contained 10% sorbitol or trehalose.        (Table 15)

In a fourth step, the outcome of the statistical analysis of the DOE wasconfirmed by a repeat of the TSA and by differential scanningcalorimetry.

Example 2.3 Solubility of the Nanobodies in Different Buffers 2.3.1Solubility in PBS and the Effect of Tween 80

During downstream processing and storage of the IL6R304, IL6R305 andIL6R306 Nanobodies in D-PBS buffer, precipitation occurred. Precipitateswere already formed during storage overnight at 5° C. or −20° C., evenin samples that were filtered (0.22 μm) before storage. This not onlyresulted in significant product loss, but also made it inconvenient toperform subsequent experiments since filtration steps needed to beincorporated constantly. From these observations it was clear that PBSis not a suitable formulation buffer for any of the IL-6R Nanobodies andthat alternative storage buffers needed to be identified. In an initialexperiment, it was assessed whether TWEEN (polysorbate) 80 could preventthis precipitation (aggregation) from occurring.

Briefly, IL6R304 (P#051108nr1) was diluted to 2 mg/mL in PBS buffer, PBSbuffer+0.1% (v:v) TWEEN (polysorbate) 80 or PBS buffer+0.2% (v:v) TWEEN(polysorbate) 80. The three samples were stored for 4 days at 5° C. andsubsequently analyzed for visible particulates (appearance testing byvisual inspection; Table 16), sub-visible particle counts (PAMAS; FIG.17), via UV spectroscopy (A320/A280 ratio, i.e. measure for the presenceof particulates; Table 16) and SE-HPLC (FIG. 18). Significantly more andlarger particles were present in the IL6R304 sample formulated in PBScompared to the IL6R304 sample formulated in PBS+TWEEN (polysorbate) 80.

2.3.2 Concentration Experiments to Determine the Solubility of IL6R304and IL6R305

IL6R304 and IL6R305, both formulated in PBS, were concentrated stepwisewith a Vivascience concentrator (VIVASPIN 500 5,000 MWCO, 500 μlconcentrator). During the concentration experiment, the retentate wasmixed gently regularly and was analyzed visually forparticulates/precipitation. The presence of insoluble aggregates in theretentate was verified by checking the protein concentration before andafter centrifugation at maximum speed. Based on the results from thethermal shift assay (see Example 2.2.1), the solubility of IL6R304 wasalso analyzed in 20 mM Histidine, pH 6.5.

In conclusion, the solubility of IL6R304 and IL6R305 in PBS was limited,with estimated values of 20 mg/mL and 15 mg/mL, respectively(concentration at which protein precipitation occurred). Noprecipitation of IL6R304 was observed at a concentration between 20-90mg/mL in L-histidine buffer suggesting that the solubility can beincreased significantly by changing the formulation buffer. Note that nosignificant protein loss has been observed.

2.3.3 Determination of the Theoretical Solubility

The theoretical solubility of IL6R304 and IL6R305 was determined usingthe PEG exclusion method in PBS buffer (IL6R304, IL6R305) or in 20 mML-histidine, pH 6.5 (IL6R304). Briefly, a concentrated Nanobody solution(30-80 mg/mL) in the respective buffer was incubated for 15 minutes atroom temperature in the presence of increasing concentrations ofPEG6000. After centrifugation at 20000×g for 3 minutes, log values ofthe [soluble protein concentration] were plotted versus PEG6000concentration (FIG. 19). By regression analysis and extrapolation to azero concentration of PEG6000, the theoretical maximum proteinconcentration (and thus solubility values) could be obtained for theNanobodies in the buffers tested. The obtained solubility valuescorrelated well with the values obtained experimentally using thestepwise concentration experiments with the VIVASPIN centrifugalconcentrators. When comparing the solubility of IL6R304 in PBS andL-histidine pH 6.5, it could be concluded that the solubility of IL6R304increased significantly in the L-histidine buffer.

Example 2.4 Storage Stability Study of the Nanobodies at 37° C.

An initial storage stability study was performed to get a generalunderstanding of the stability of the IL-6R Nanobodies and to determineif adding mannitol in the formulation buffer has a beneficial effect inminimizing the formation of potential dimers, as was observed forRANKL008a (see Example 1.3).

The three IL-6R Nanobodies were formulated in different buffers (Table17) at a concentration of 10 mg/mL (IL6R304), 7.1 mg/mL (IL6R305) and10.3 mg/mL (IL6R306). The stability of the different samples wasassessed in accelerated stress conditions at 37° C. Samples wereanalyzed after 1 week using SE-HPLC and RP-HPLC. Selected samples ofIL6R304 and IL6R305 were also analyzed after 3 weeks of storage.

2.4.1 SE-HPLC Analysis

For all three IL-6R Nanobodies, prolonged storage at 37° C. resulted inthe formation of prepeaks and some minor postpeaks. The postpeaksprobably corresponded to degradation products (due to remainingproteolytic activity in sample). The surface area of these postpeaksremained very low, suggesting only minimal degradation after 3 weeks at37° C.

All three IL-6R Nanobodies had a strong tendency to formdimers/oligomers (aggregates), which were visible as prepeak(s) in thechromatograms of the SE-HPLC analysis. An example chromatogram is shownin FIG. 20. The peak area of the prepeak increased significantly overtime (represented as % aggregates in FIG. 21 and FIG. 22) and wasaccompanied by a concomitant decrease in surface area of the main peak.The propensity to form dimmers/oligomers appeared to be somewhat higherfor IL6R305 than for IL6R304. Also, more dimmers/oligomers were beingformed in PBS compared to the other buffers that were tested.Importantly, the lowest amounts of oligomers were observed in themannitol-containing formulations.

2.4.2 RP-HPLC Analysis

The RP-HPLC profile of IL6R304 at time point 0 weeks included a mainpeak with a shoulder eluting before the main material and a postpeakthat was not well resolved from the main peak. This postpeak most likelycorresponded to the pyroglutamate-containing variant of IL6R304. Thesurface area of this peak increased with storage time and was highest inPBS and phosphate buffer compared to the acetate and histidine buffers.After 3 weeks of storage, the surface areas of two thus far unidentifiedprepeaks increased (FIG. 23).

The RP-HPLC profile of IL6R305 at time point 0 weeks included a mainpeak with some minor prepeaks. The resolving power of the RP-HPLC methodused was insufficient to separate the pyroglutamate-containing variantfrom the main material.

Example 2.5 Osmolality Measurement

According to the European Pharmacopoeia, a solution is consideredisotonic if it has an osmolality of 290±30 mOsm/kg. Osmolalitymeasurements on 20 mM L-histidine pH 6.5 containing differentconcentrations of excipients were therefore performed to define therange of excipient concentration that would be acceptable for anisotonic liquid formulation of IL6R304.

IL6R304 (10 mg/mL) was formulated in the different buffers of Table 18.The results from osmolality measurement of these formulations are shownin FIG. 24.

Example 2.6 Storage Stability Study of 16R304 at 5° C. and 37° C.

An overview of the different formulation buffers and methods used instability testing of IL6R304 batch P#051108nr1 is given in Table 18 andTable 19, respectively. Because IL6R304 was found to be prone toaggregation and precipitation, TWEEN (polysorbate) 80 was added to mostformulations.

2.6.1 Appearance and OD280

No turbidity was observed in the samples stored for 5 weeks at 5° C.,indicating that 20 mM L-histidine pH 6.5 is a much better storage bufferfor IL6R304 than PBS.

After storage for 1 week at 37° C., a slight turbidity was observed inall 12 samples. In the IL6R304 samples in buffers 1, 2, 3, 7, 8 and 9more opalescence was observed compared to IL6R304 in the buffers 4, 5,6, 10, 11 and 12, which all contained mannitol. After storage for 2weeks at 37° C., slightly more turbidity was observed compared to the 1week samples. However, the trend observed for the 1 week samplescontinued: the IL6R304 samples in buffers 1, 2, 3, 7, 8 and 9 showedmore opalescence compared to the mannitol-containing buffers. Afterstorage for 5 weeks at 37° C., turbidity was still present and slightlyless in the samples containing mannitol. However, it seemed like theturbidity had not increased compared to the 2 weeks samples.

Despite the opacity observed in the stressed samples, the proteinconcentration in the samples has not decreased significantly (data notshown) although there was a slight trend to a lower concentration due toa higher turbidity. Also, the OD320/OD280 ratio, which is a measure forturbidity or the presence of particulates, was <0.05 in all bufferconditions. In fact, the ratio was 2-10 fold lower than observed in theunstressed sample in PBS, again showing that the L-histidine pH 6.5buffer has a stabilizing effect on IL6R304.

2.6.2 SE-HPLC Analysis

Samples of the reference material (0 weeks) and samples stored for up to6 months at 5° C. and 37° C. were analyzed using SE-HPLC.

No differences were observed between the SE-HPLC profiles of thereference samples (at 0 weeks) and the samples stored for up to 5 weeksat 5° C. In addition, there were no significant differences between thedifferent buffers. The small amounts of aggregates already present inthe start material were not increasing with prolonged storage time (FIG.25(B)), indicating that the Histidine buffer had a stabilizing effect onILR304, even in the absence of excipients such as TWEEN (polysorbate)80, mannitol or sucrose. Note that IL6R304 formed aggregates when storedfor a short time (hours-days) at 5° C. in D-PBS buffer.

SE-HPLC analysis of the samples stored for 6 months at 5° C. also didnot show increase in area % of the prepeaks, meaning that no oligomerswere formed under these storage conditions, not even in the formulationcontaining only 20 mM L-histidine, pH 6.5 i.e. without TWEEN(polysorbate)-80 or any excipient (data not shown).

Prolonged storage at 37° C. resulted in the formation of prepeaks andsome minor postpeaks. The postpeaks probably corresponded to degradationproducts (due to remaining proteolytic activity in sample). The relativearea (%) of these peaks increased only slightly, implying thatdegradation was restricted to a minimum. The other peaks visible in thechromatograms were background peaks arising from the buffer components.

The peak area of the prepeaks increased significantly over time (FIG. 25(A) and FIG. 26 (B)). Given the relative position of the prepeaks to themain peak, the prepeaks most likely represented dimeric or oligomericforms (aggregates) of IL6R304. The peak surface area of the prepeakincreased with storage time and was accompanied by a concomitantdecrease in surface area of the main peak.

An important observation was that the propensity to formdimers/oligomers was buffer-dependent: the propensity to oligomerize wassignificantly lower in the mannitol- and sucrose-containingformulations. Glycine appeared not to have such a positive effect inpreventing the oligomerization process. TWEEN (polysorbate) 80 had noinhibitory effect on the formation of oligomers.

Importantly, the % oligomers observed in all 12 L-histidine buffersafter storage for 3 weeks at 37° C. was significantly lower than theequivalent sample in D-PBS buffer, i.e. 2.2-4.6% in L-histidine, pH 6.5compared to 11.7% PBS (FIG. 26(A)). This buffer-dependent effect on thephysical stability of IL6R304 correlated very well with thebuffer-dependent differences observed in thermal stability testing ofIL6R304 (Example 2.2): the melting temperature of IL6R304 was found tobe only 58.8° C. in PBS but is 62.8° C. in 20 mM L-histidine, pH 6.5.Increasing the intrinsic stability of IL6R304 by changing theformulation buffer from PBS to L-histidine proved to have a clearbeneficial effect on its stability upon storage.

In the samples stored for 6 months at 37° C., the lowest % of oligomerswas found in the formulation containing 10% sucrose, again corroboratingthe stabilizing effect of sucrose on IL6R304 (Table 20).

2.6.3 RP-HPLC Analysis

Samples of the reference material (0 weeks) and samples stored for up to5 weeks at 5° C. and 37° C. were analyzed using RP-HPLC.

The RP-HPLC profiles at time point 0 weeks included a main peak, twopre-peaks and a badly resolved postpeak. This post-peak most probablycorresponded to the pyroglutamate-containing variant of IL6R304.

The RP-HPLC profiles of the reference batch and the stability samplesstored for up to 5 weeks at 5° C. were found to be comparable.

In the stability samples stored at 37° C., the peak surface area of thepyroglutamate peak increased with storage time while the surface area ofthe main peak decreased. The total area remained unchanged.

After 5 weeks of storage at 37° C., the surface area of the two prepeakshad increased in all buffer conditions. The identity of these variantsis unknown at the moment, but could correspond to degradation fragments.

There were no significant buffer-dependent differences in the RP-HPLCprofiles of the different samples suggesting that chemicalmodifications, such as pyroglutamate formation and oxidation which aretypically detected by RP-HPLC, were limited and at present unaffected bythe buffer.

Example 2.7 Storage Stability Study of IL6R304 at −70° C., −20° C., 5°C., 25° C. and 37° C.

IL6R304 was formulated at 10 mg/mL in the 10 different buffers shown inTable 21, stored at −70° C., −20° C., +5° C. and +37° C. for 8 weeks andfor 1 week +25° C. Stability samples were analyzed using SE-HPLC,RP-HPLC, OD280 and visual inspection. Selected samples were alsoanalyzed using BIACORE (HSA binding) and potency assays (HSA and IL-6R).

2.7.1. Storage for 8 Weeks at −70° C., −20° C., 5° C. and 1 Week at 25°C.

IL6R304 was shown to be stable after storage for 8 weeks at −70° C.,−20° C., 5° C. and for 1 week at 25° C. in all 10 buffers tested. Nosignificant differences were observed in potency, turbidity, SE-HPLC andRP-HPLC profiles between the reference material and the 10 differentstorage samples.

2.7.2. Storage for 8 Weeks at 37° C. Appearance and OD280

Compared to the samples stored at −70° C., −20° C. and 5° C., turbiditywas observed in the samples stored at 37° C. The absorbance values at350 nm had increased accordingly to >0.01 AU in most buffers, althoughthe A350/A280 ratio was still <0.05 in all buffer conditions. Despitethe opacity observed in the stressed samples, the protein concentrationin the samples had not decreased significantly.

SE-HPLC

Prolonged storage at 37° C. resulted in the time-dependent formation ofa postpeak and prepeak. The postpeak has a retention time between 22 and23 minutes and most likely corresponded to IL6R304 degradationfragments. The surface area of this peak however remained low(approximately 2%), suggesting only minimal degradation after 8 weeks at37° C. The other postpeaks visible in the chromatograms were backgroundpeaks arising from the buffer components.

The SE-HPLC profile of IL6R304 at time point 0 weeks included a mainpeak and two minor prepeaks, which were not completelybaseline-resolved. The surface area of the prepeaks increased over timeand was accompanied by a concomitant decrease in surface area of themain peak. Given the relative position and heterogeneity of theprepeaks, they most likely represented dimeric and/or oligomeric formsof IL6R304. Because of this heterogeneity and the decreasing resolutionbetween the prepeaks over time, the peaks were for simplicity integratedas a single peak.

An important observation was that the propensity to formdimers/oligomers was buffer-dependent: about 2-fold less oligomers werebeing formed in L-histidine buffer compared to phosphate buffer (FIG.27, FIG. 28). The lowest amount of oligomers was observed in thetrehalose-containing formulation, followed by the sucrose-containingformulation. Overall, after storage of IL6R304 at 37° C. for severalweeks, the amount of oligomers present in these buffers wassignificantly less than observed previously in D-PBS or in L-histidinepH 6.5 devoid of any excipient.

The presence of a non-reducing sugar suppressed the extent of IL6R304oligomerization considerably.

RP-HPLC

The RP-HPLC chromatograms from the ILR304 stability samples stored forup to 8 weeks at 37° C. are shown in FIG. 29.

The RP-HPLC profile of ILR304 at time point 0 weeks included a mainpeak, with 2 badly resolved shoulders eluting before the main material,a first postpeak corresponding to the pyroglutamate-containing variantof ILR304 and a second postpeak, corresponding to the ILR304 variantmissing one disulphide bridge. The identity of both variants has beenconfirmed by LC-MS.

After storage during 1 week, the surface area of the second postpeak haddecreased to a relative area % of 0, most likely due to spontaneousoxidation into the correctly folded molecule.

The surface area of the pyroglutamate peak increased with storage timewhile the surface area of the main peak decreased. The total surfacearea was not changing significantly over time or among the differentbuffers. FIG. 30 clearly demonstrates that the kinetics of pyroglutamateformation was different in L-histidine, pH 6.5 versus phosphate, pH 6.5.At all time points, less pyroglutamate was present in the L-histidinebuffer. On the other hand, there was no correlation between the type ofexcipient present in the buffer and the amount of pyroglutamate beingobserved.

After storage for 8 weeks, two new postpeaks were being formed. Thefirst postpeak was situated between the main peak and the pyroglutamatepeak, while the second postpeak eluted just after the pyroglutamatepeak. The identity of these variants is currently not known.

Capillary Isoelectric Focusing (cIEF)

cIEF integration data of IL6R304 stored for 8 weeks at 37° C. in thedifferent buffers are shown in Table 22. The samples formulated in 15 mML-histidine, pH 6.5 contain less charge variants compared to thephosphate buffer.

Potency Assay and BIACORE

The potency of the samples stored for 8 weeks at 37° C. in buffers 1-5was determined relative to an unstressed reference batch using theHSA-binding ELISA and the inhibition ELISA for IL-6R as described inExamples 2.1.8 and 2.1.9 (Table 23). The HSA binding functionality ofthe samples stored in buffers 1-10 was also analyzed using BIACORE(Table 24). Samples formulated in the same buffers and stored at −70° C.were included as the reference molecules.

Whereas the potency assays showed comparable HSA and IL-6R bindingpotencies between the stability samples and the reference material,BIACORE analysis demonstrated some differences in HSA bindingactivities.

Overall, the activities of the samples formulated in phosphate buffer(buffers 6-10) were lower than in L-histidine (buffers 1-5). Afunctionality loss of approximately 16% was observed in the bufferscontaining a combination of sucrose and glycine (buffer 4 and 9). Thecombination of sucrose and mannitol (buffer 5 and 10) showed no loss offunctionality of IL6R304 in the L-histidine buffer, while a decrease of10% was observed in the phosphate buffer. Formulations containing eithermannitol, sucrose or trehalose showed an activity between 90 and 100%after storage for 8 weeks at 37° C.

General Conclusion about the Storage Stability Study at 37° C.

Storage for up to 8 weeks of IL6R304 in different formulation buffersunder temperature stress conditions (37° C.) resulted in the followingobservations:

-   -   The propensity of IL6R304 to form oligomers was dependent on the        buffer and excipient: about 2-fold less oligomers were being        formed in L-histidine buffer compared to phosphate buffer, while        the presence of a non-reducing sugar suppressed the extent of        IL6R304 oligomerization even further;    -   The chemical stability of IL6R304 was better in L-histidine        buffer compared to phosphate buffer;    -   The HSA binding activity was maintained longer in L-histidine        buffer compared to phosphate buffer.

Example 2.8 Stability Under Stir Stress

IL6R304 was formulated at 1 mg/mL in the 10 different buffers shown inTable 21. Aliquots of 5 mL were stirred at maximum speed for up to 24hours at 2-8° C. Samples were analyzed after 2, 4 and 24 hours ofstirring.

All solutions remained clear after 2 hours of stirring (Table 25). Anincrease in turbidity was observed in six out of ten buffers after 4hours. The highest opalescence was present in buffer 10. Overall, theincrease in turbidity was more pronounced in the phosphate buffers(buffer 6-10). These observations were confirmed after determination ofthe aggregation index, defined as 100*OD350/(OD280-OD350) (FIG. 31). Nosoluble aggregates were observed during SE-HPLC of the differentsamples.

In conclusion, the stir stress data suggest a somewhat better stirstress stability of IL6R304 in L-histidine, pH 6.5 compared to phosphatebuffer, pH 6.5. No significant differences were observed between thevarious excipients, although a slightly higher turbidity was observed inthe presence of 10% trehalose and 2.5% mannitol/5% sucrose.

Example 2.9 Long-Term Stability Study at −70° C., +5° C. and +25° C.

IL6R304 batch CMC-D-0048, formulated in 15 mM L-Histidine, 8% sucrose,0.01% TWEEN (polysorbate) 80 (pH6.5) at 10.52 mg/mL, was stored for 6months at −70° C., +5° C. and +25° C. Samples were analysed after 3 and6 months of storage by visual inspection (appearance), A280 (content),SEC-HPLC, cIEF, RP-HPLC and potency assays (IL6R inhibition assay andHSA binding assay). The results are summarized in Table 42, Table 43 andTable 44 for storage at −70° C., +5° C., and +25° C., respectively.

There were no significant changes in appearance, content, potency, cIEFand HPLC profiles between the control sample (timepoint 0 months) andall test samples stored at −70° C. or 5° C. indicating that IL6R304 isstable for at least 6 months under these conditions.

Regarding the sample stored at +25° C., the following observations weremade when comparing the results of the stressed samples and the controlsample:

-   -   SE-HPLC: there is a small, yet gradual increase in the surface        area of the pre peak (oligomers) and post peak (degradation        fragments).    -   cIEF: a post peak is being formed which is believed to        correspond to the pyroglutamate variant.    -   RP-HPLC: three new peaks are being formed, i.e. a pre peak, most        likely corresponding to degradation fragments that are present        in the samples (see also SEC-HPLC data) and two yet unidentified        post peaks. The surface area of pre peak 2 and post peak 2        (pyroglutamate) are gradually increasing with prolonged        incubation time.    -   No potency loss is observed in the samples stored for up to 6        months at +25° C.

Example 3 Formulation and Stability Studies with Nanobodies that BindIL23 Example 3.1 Materials and Methods Used in the Study 3.1.1 SingleVariable Domains

23IL0064 (SEQ ID NO: 5;EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVKGRFTISRDNSKKTLYLQMNSLRPEDTAVYYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAMGWFRQAPGKGREFVSRISQGGTAIYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAKDPSPYYRGSAYLLSGSYDSWGQGTLVTVSS) has been described as SEQ ID NO: 2616 in WO2009/068627. 23IL0064 consists of three humanized variable domains of aheavy-chain llama antibody: 119A3v16 and 81A12v4, binding differentepitopes of IL23 p19, and the ALB8 binding HSA. 23IL0075 (SEQ ID NO: 6;EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVKGRFTISRDNSKKTLYLQMNSLRPEDTAVYYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAMGWFRQAPGKGREFVARISQGGTAIYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAKDPSPYYRGSAYLLSGSYDSWGQGTLVTVSS) has been described as SEQ ID NO: 2622 in WO 2009/068627.23IL0075 consists of three humanized variable domains of a heavy-chainllama antibody: 119A3v16 and 81A12v5, binding different epitopes of IL23p19, and the ALB8 binding HSA. The subunits in both Nanobodies are fusedhead-to-tail with a 9G/S linker.

23IL0064 (3.79 mg/mL in D-PBS) and 23IL0075 (4.21 mg/mL in D-PBS) wereexpressed in Pichia pastoris. After clarifying the fermentation brothvia a centrifugation step, followed by a TFF step, the Nanobodies werecaptured on MABCAPTURE A (Poros), followed by an elution at pH 2.6 using100 mM glycine. A buffer switch to 1/10 D-PBS was performed, and theNanobodies were further polished on Poros 50HS (Poros). Finally, atreatment with 50 mM OGP for LPS-removal was performed, followed by afinal size exclusion step using SUPERDEX 75 pg (GE Healthcare).

Unstressed samples in D-PBS or other formulations were used as referencematerial for analyzing the storage stability samples.

3.1.2 Other Critical Reagents

Reagents used in the study are given in Table 26. The completecomposition of D-PBS was 137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphatedibasic, 2 mM Potassium Phosphate monobasic.

3.1.3 Equipment and Methods for Measurements

HPLC experiments were carried out on an Agilent 1200 series instrumentfrom Agilent Technologies (Palo Alto, USA). The columns used were:

-   -   RP-HPLC: ZORBAX 300SB-C3 5-micron, 4.6×150 mm (Agilent, Cat. No.        883995-909) and ZORBAX 300SB-C8 5-micron, 4.6×150 mm (Agilent,        Cat. No. 883995-906)    -   SE-HPLC: TSK-GEL G2000SW_(XL) (Tosoh Bioscience, Japan;        Part#08540)    -   IEX-HPLC: PROPAC WCX-10, 4×250 mm, 10 μm (Dionex)

Concentration determinations of the Nanobodies was done with NANODROPND-1000 (Thermoscientific), with a Uvikon 943 Spectrophotometer (KontronInstruments) or an Eppendorf Biophotometer 6131 at 280 nm.

Particle size distribution was measured on a PAMAS SVSS-C particlecounter (PArtikelMess-und AnalyseSysteme GMBH).

Osmolality measurement was done with an osmometer Model 3320 fromAdvanced instruments.

The thermal shift assay was performed on a UghtCycler480 Q-PCR device(Roche).

For determination of the Tm, an automated VP-capillary DifferentialScanning Calorimeter (DSC, MicroCal) was used.

Elastic light scattering was measured in a Jasco Spectrofluorometer(FP-6500).

3.1.4 Purity Assay of the Nanobodies by Size Exclusion High PerformanceLiquid Chromatography (SE-HPLC)

The SE-HPLC assay consisted of a pre-packed silica gel TSK-GELG2000SW_(XL) column equipped with a guard column pre-column filter, amobile phase consisting of KCl, NaCl and phosphate buffer pH 7.2 (D-PBS)and UV detection at 280 nm. The relative amount of specific proteinimpurity was expressed as area %, and was calculated by dividing thepeak area corresponding to the specific protein impurity by the totalintegrated area.

3.1.5 Purity Assay of the Nanobodies by Reverses Phase High PerformanceLiquid Chromatography (RP-HPLC)

In the RP-HPLC assay a ZORBAX 300SB-C3 column or ZORBAX 3005B-C8 column(Agilent Technologies, Palo Alto, US) was used. The relative amount of aspecific protein impurity was determined by measuring the lightabsorbance of the components eluting from the RP-HPLC column. Therelative amount of a specific protein impurity, expressed as area %, wascalculated by dividing the peak area corresponding to the impurity bythe total integrated area.

3.1.6 Purity Assay of the Nanobodies by Ion Exchange High PerformanceLiquid Chromatography (IEX-HPLC)

The IEX-HPLC assay combined the use of a pre-packed Dionex PROPAC WCX-10weak cation exchange column, a mobile phase consisting of citrate bufferpH5.5 and UV detection at 280 nm. After loading the protein(s) on thecolumn, bound materials were eluted by a sodium chloride gradient. Therelative amount of the specific protein, variant, or impuritiesexpressed as area %, was calculated by dividing the peak areacorresponding to the specific protein or to any protein impurity by thetotal area of all integrated peaks.

3.1.7 Measurement of Particle Size Distribution (PAMAS)

The measurements on the PAMAS SVSS-C particle counter were performed asfollows: 100 μl sample was diluted 1/10 in 1 mL MILLI-Q water and 10consecutive measurements were performed in all 16 channels (diameter set1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100, 150 and 200 μm). Forcalculation of the average value, the first 2 measurements were excludedand the dilution factor was taken into account. The results are given ascumulative data (total particle counts>x μm) or differential data (totalparticle counts between diameter x and y μm). Only the cumulative dataare presented.

3.1.8 Capillary Isoelectric Focusing (cIEF)

Capillary Isoelectric Focusing (cIEF) is an analysis/separationtechnique that differentiates proteins with respect to charge, i.e., itseparates proteins according to their isoelectric points (pI). Theseparation principle is similar to gel-based/flatbed IEF but differsmainly in its format, that is, the separation takes place in an opentube of narrow format (capillary) eliminating the need for anyanticonvective matrix support. Also, cIEF is a fully automatedinstrument with online detection and data acquisition. A drawback oftraditional cIEF in a conventional CE instrument is that the focused(stationary) zones must be mobilized past the single-point detectionarea in order to record the signal. During mobilization the zones maybecome broadened with contaminant loss of resolution and decreaseddetectability. Moreover, the analysis time and risk of proteinaggregation/precipitation will increase. By imaging cIEF the focusingprocess is followed in real-time over the whole-column/capillary by aCCD camera excluding the mobilization step. As soon as the focusingprocess is completed the analysis run is finished.

Example 3.2 Melting Temperature of the Nanobodies

The measurement of the melting temperature of a protein in differentbuffers is a faster way to screen for buffers in which the protein hasthe highest physical stability. It is generally accepted that this willbe predictive for the long term stability at lower temperatures. Themelting temperature can be determined using many different techniques.

3.2.1 Melting Temperature of 23IL0064 in Different Formulation BuffersMeasured by Thermal Shift Assay

We used the thermal shift assay (TSA), which measures the change influorescence intensity when the SYPRO orange binds the hydrophobic partsof the protein that is undergoing thermal unfolding. The thermal shiftassay was performed on the LightCycler480 Q-PCR device (Roche) makinguse of 96-well plates.

In a first study, the effect of buffer couple, ionic strength and pH onthe thermodynamic stability of 23IL0064 was evaluated in a thermal shiftanalysis experiment. The tested buffers used are presented in Table 28.Each buffer was tested at a pH interval spanning 1 pH unit around theirpKa. An overview of the results is presented in FIG. 32. The meltingtemperatures decreased with increasing salt concentration. Further pH5.2 and pH 5.5 seemed less favorable compared to pH 6.2 and above. Thebest buffers were Hepes (pH 7 and 8) and Histidine pH 6.5. In allbuffers, the addition of mannitol slightly increased the meltingtemperatures.

3.2.2 Melting Temperature of 23IL0064 and 23IL0075 in DifferentFormulation Buffers Measured by Thermal Shift Assay and DifferentialScanning Calorimetry (DSC)

23IL0064 and 23IL0075 were analyzed in parallel in different formulationand purification buffers by the thermal shift assay (TSA) (Tables 34)and DSC (Table 33). For both molecules and in all tested buffers(acetate, MES, Hepes, TRIS, and Histidine), the melting temperaturesdecreased with increasing NaCl concentration, and the meltingtemperatures were highest in the presence of mannitol.

3.2.3 Melting Temperature of 23IL0075 in a Wide Range of FormulationBuffers Measured by Thermal Shift Assay

The melting temperature for 23IL0075 in another range of buffers and alower pH range was screened by TSA. For this, a design of experimentswas set up to investigate the Tm of 23IL0075 varying the followingparameters: pH range between 5 and 6, buffer concentration between 10and 50 mM, and 4 different buffers: histidine, acetate, phosphate andsuccinate. A full factorial design+central composite design wasperformed, in total 22 different buffers were tested in 84 experiments.The experiments were divided over two 96-well plates, on the secondplate the pH range was expanded to 4.8 and 6.2, and the bufferconcentration to 7 and 65 mM. The phosphate buffer was only tested at pH6 and 6.2. From the experimental results, for each buffer a model wasbuilt that was used to predict Tm's as a function of the bufferconcentration and the pH. The results are shown in Table 35.

Succinate clearly had overall the lowest predicted Tm values. Foracetate and histidine at low concentrations and high pH the largestpredicted Tm values were obtained. In fact the model showed for allbuffers highest predicted Tm values for lowest buffer concentrations.According to the Design-Expert Numerical Optimization of the model thebest buffer was histidine buffer pH 6.2 (desirability 1), followedclosely by acetate pH 6 (desirability 0.96) and phosphate pH 6(desirability 0.90).

3.2.4 Confirmation of Tm by Differential Scanning Calorimetry

We confirmed the Tm determination by differential scanning calorimetryin the buffers selected in Example 3.2.3. The results are shown in thegraph in FIG. 33. The trends were identical to what was observed in theTSA though the absolute values of the Tm were higher: around 62° C. inDSC compared to around 59° C. in the TSA.

3.2.5 Melting Temperature of 23IL0075 in a Range of Formulation Buffersto Explore a Wide Range of Excipients Measured by Thermal Shift Assay

A second design of experiments (DOE) was prepared to study the influenceof some combinations of excipients on the thermodynamic stability of23IL0075. The combination of a sugar or a polyol (mannitol, sucrose orsorbitol), with an amino acid (Glycine or arginine/glutamic acidmixture), and a non-ionic detergent (TWEEN (polysorbate) 80, TWEEN(polysorbate) 20 or P-F68) were studied. These combinations were testedin three buffers (10 mM Acetate pH 5.5, 10 mM Histidine pH 6.0 and 10 mMPhosphate pH 6.0) (see Table 35). Based on the measured meltingtemperatures, the optimal buffer compositions were calculated (see Table36). Highest melting temperatures were predicted for the histidinebuffer, the lowest melting temperatures for the phosphate buffer. Forall three buffers sucrose came out as the best excipient, the mixture ofarginine and glutamic acid as the worst. Mannitol and sorbitol, alone orin combination with glycine were also good as excipient, though alwaysgave a little bit lower Tm than sucrose. Glycine alone was not soefficient.

Example 3.3 Solubility of the Nanobodies 3.3.1 Concentration Experimentsto Determine the Solubility of 23IL0064

The IL23 Nanobody 23IL0064, respectively in D-PBS buffer, in NaPhosphate10 mM pH 7 with 50 mM NaCl, and in L-histidine 40 mM pH 6 with 50 mMNaCl, was concentrated stepwise with a VIVASPIN concentrator(Vivascience 5,000 MWCO 0.5-mL and 5-mL concentrators). During theconcentration experiment, the retentate was regularly gently mixed andthe concentration determined by OD280 measurements. The presence ofinsoluble aggregates in the retentate was verified by checking theprotein concentration before and after centrifugation at maximum speedin a benchtop Eppendorf centrifuge. In all three buffers a concentrationof 50 mg/mL could be reached without visual precipitation. Then theprotein solution was transferred from the 5 mL to the 0.5 mL VIVASPIN®concentrator and concentrated at a much higher centrifugal force(15000×g instead of 1500×g). This caused rapid further concentration,and local concentrations close to the membrane were even much higherthan the average concentration measured after recovery of the retentate.In Table 27, the final concentrations achieved are summarized, obtainedafter pipetting up and down the retentate, and centrifugation to removeprecipitate.

All samples were also analyzed by SE-HPLC. Concentrating in D-PBS bufferresulted in an increase in % pre-peak. In the 10 mM phosphate buffer andin the 40 mM Histidine buffer, both with 50 mM NaCl, the SE-HPLC profileremained exactly the same at 83 and 150 mg/mL compared to the startingmaterial (FIG. 34). A generic SE-HPLC method was used on a PhenomenexBIOSEP SEC 5-2000 column, with D-PBS as mobile phase at 0.2 mL/min.

A sample of the concentrated 23IL0064 protein solutions was diluted to55 mg/mL using the respective buffers for further use in the PEGprecipitation method.

3.3.2 Determination of the Theoretical Solubility of 23IL0064

The theoretical solubility of 23IL0064 was determined using the PEGexclusion method in D-PBS buffer, in NaPhosphate 10 mM pH 7, NaCl 50 mM,and in L-histidine 40 mM pH 6, NaCl 50 mM. Briefly, a concentratedNanobody solution (55 mg/mL) in the respective buffer was incubated for15 minutes at room temperature in the presence of increasingconcentrations of PEG6000. After centrifugation at 20000×g for 3minutes, log values of the [soluble protein concentration] were plottedversus PEG6000 concentration (FIG. 35). By regression analysis andextrapolation to a zero concentration of PEG6000, the theoreticalmaximum protein concentration (and thus solubility values) could beobtained for the Nanobody in the buffers tested.

When extrapolating from the regression plots to a zero concentration ofPEG6000, a theoretical solubility value of 60 mg/mL and of 288 mg/mL wascalculated for 23IL0064 in D-PBS and in 10 mM phosphate buffer/50 mMNaCl respectively. 23IL0064 showed extremely good solubility in thehistidine buffer: in the experiment starting from 55 mg/mL only at a PEGconcentration of 27% the protein started to precipitate slightly.Therefore the experiment was repeated with the protein dissolved at 150mg/mL. There only at 10% PEG precipitation occurred, but then no volumewas left for OD measurements. So the solubility was actually too high toobtain a value in this assay.

In conclusion highest solubility was obtained in 40 mM Histidine pH 6with 50 mM NaCl. In phosphate buffer with 50 mM NaCl, the solubility wasbetter than in D-PBS (with 137 mM NaCl).

3.3.3 Determination of the Theoretical Solubility of 23IL0064 and23IL0075

The theoretical solubility was determined for 23IL0064 and 23IL0075using the PEG exclusion method in NaPhosphate 10 mM pH7, NaCl 50 mM, andin L-histidine 40 mM pH 6, NaCl 50 mM (the same buffers as used in theprevious solubility study for 23IL0064 described above). The graphs(FIG. 36 and FIG. 37) representing the protein concentration in thesupernatant as a function of the % PEG concentration were very similaras obtained in the previous experiment. Again the apparent solubility inhistidine was higher than in the phosphate buffer, and could not becalculated due to minimal precipitation under the protein (5 mg/mL) andPEG concentrations (26.7%) used. The calculated solubility in phosphatebuffer pH 7 for 23IL0064 and 23IL0075 were lower than in the previousexperiment, i.e. 55 and 42 mg/mL respectively, while we observed up to288 mg/mL in the earlier experiment. We must stress though that theseexperiments were performed pipetting extremely low volumes of highlyviscous solutions, and therefore the absolute numbers of solubilityshould be confirmed with other techniques.

Example 3.4 Stressed Stability Studies for 23IL0064 in D-PBS

An initial 37° C. stressed stability study was performed in D-PBS. Theoriginal batch was sterilized through a 0.22 μm filter and 500 μl wasstored at 37° C. in 1.5 mL-eppendorf vials for each time point (4, 8,12, 16, 20 and 24 weeks). Additionally approximately 9×100 μl was storedat −20° C. (reference). A first sample was already analyzed after 3weeks using SE-HPLC and a pre-peak of aggregates was detected (3%) (FIG.38). After 4 weeks at 37° C., the total peak area on SE-HPLC and onRP-HPLC was reduced to only half of the reference sample (FIGS. 38 and39). We therefore decided to prematurely terminate the stability study.In some of the remaining samples the content was still measured by OD280after centrifugation. The loss of material in the 4w-37° C.-samplethrough precipitation (all samples were centrifuged before analysis) wasconfirmed in the additional 3 samples stressed for 6 weeks at 37° C.: in2 of the 3 samples half of the material was lost (Table 29).

It can be concluded that 23IL0064 in D-PBS easily formed aggregateswhich precipitate following storage for 4 weeks on at 37° C. In theRP-HPLC analysis of the 37° C.-stressed sample also 12% post-peak wasobserved corresponding to N-terminal pyro-glutamate formation (FIG. 41).

Example 3.5 Stressed Stability Study for 23IL0064 in Histidine Buffer

From the results described in previous Examples on the solubility, thethermal shift assay, and the 37° C.-stressed stability in D-PBS for23IL0064, it was concluded that phosphate was not the optimal buffer forformulation of 23IL0064. In the TSA described in Example 3.2.2, weexplored some potential formulation buffers and the highest Tm's wereobtained in histidine pH 6.5, Hepes pH 7, and Hepes pH 8. In thesolubility experiment (see Example 3.3) the solubility in a 40 mMhistidine pH 6, with 50 mM NaCl was very high. We therefore decided totest the storage stability in histidine buffer. In Table 30 a list oftested formulation buffers is given. The goal of this set-up was tocompare histidine pH 6.5 with histidine pH 6, investigate the influenceof some commonly used excipients, and the influence of a higherconcentration on the stability (difference between 5 mg/mL and 22mg/mL). One sample in Hepes pH 8 was also included, only to be testedafter 3 weeks at 37° C.

For this study, 3.2 mL of the original batch was dialyzed to the 20 mMHepes buffer pH 8 and approximately 65 mL (approx. 246 g) was dialyzedto the 20 mM Histidine buffer pH 6.5. The excipients were added inconcentrated solutions (2×), and the sample at pH 6 was prepared byadding HCl. The samples were then concentrated to approximately 5 mg/mL,sterilized through a 0.22 μm filter and aliquoted in 1.5 mL-eppendorfs(500 μL/eppendorf) for storage under the different conditions.

3.5.1 Stability During Freeze Thaw

One sample of each of above formulations in histidine was subjected to10 freeze/thaw cycles. The samples were analyzed by RP-HPLC and OD280content. No difference with the reference sample (one freeze/thaw) wasobserved.

3.5.2 Shear Stress

Two samples of each of above formulations in histidine were subjected toshear stress. The test was conducted in a cold room (4-8° C.) in smallglass tubes with 300 pI of protein solution, stirred through a magneticbar for 4 and 8 hours at a medium rotation speed.

In all samples stressed with 4 and 8 hours of shearing clear opalescencewas present. This was quantified by OD280 content analysis aftercentrifugation of the samples. Analysis by SE-HPLC did not reveal anyaggregates. RP-HPLC analysis after 4 hours of shear stress showed nodegradation, but after 8 hours of stress some increase of thenon-resolved pre-peak appeared, especially in the concentrated sample.Also on SDS-PAGE generally no degradation was detected. In Table 31 acrude ranking based on the opalescence and material loss in the contentanalysis is given.

3.5.3 Storage at 4° C., 25° C. and 37° C. for 6 Weeks

The first analysis was performed after 2.5 weeks at 25° C. and 37° C.storage. The samples were analyzed on RP-HPLC (see also Table 32),SE-HPLC, SDS-PAGE and OD 280/350. Very little degradation was observedafter 2.5 weeks (data not shown). The results after 6 weeks of storageat 37° C. are discussed below (for the sample in Hepes pH 8, the 2.5weeks 37° C. results are discussed).

RP-HPLC Analysis

RP-HPLC analysis was performed mainly to detect chemical degradation.Stress at 25° C. and at 37° C. typically caused increase of thepost-peak corresponding to the N-terminal pyroglutamate. This post-peakincreased less in the histidine buffer pH 6 than in pH 6.5, and fastestin the Hepes buffer pH 8: e.g. after 2.5 weeks at 37° C. there was 11%pyroglutamate post peak in Hepes pH 8, 7% in histidine pH 6.5, and 5% inhistidine pH 6 (data not shown).

Further a second unknown post-peak appeared. In FIG. 40 an overview ofthe integration data is given. In FIG. 41 an overlay between thechromatograms obtained for the different storage temperatures of23IL0064 in histidine buffer pH 6.5 at 22 mg/mL is given.

SE-HPLC Analysis

For the samples at 5 mg/mL stressed for 6 weeks at 37° C., small amountsof aggregates (between 0.5 and 1%) were observed. The peaks wereintegrated. The separated pre- and postpeaks were never higher than 1percent.

For the sample at 22.4 mg/mL however, 3% aggregates were detected in thesample stored at 37° C.

SDS-PAGE Analysis

Analysis by SDS-PAGE showed little degradation. For the samples stressedat 37° C. for 6 weeks, a slight increase in intensity of a degradationband at a Mw of approximately 27 kDa was observed and some thin bandsunder the main band were present (FIG. 42). No difference between thedifferent formulation buffers was observed.

Analysis of OD280, OD350 and Subvisible Particles

All samples in the storage stability study were further analyzed fortheir Nanobody content (by OD280), for their opacity (by OD350), and todetect subvisible particles (by PAMAS). Very similar results wereobtained between the reference and the different temperature storagesamples (data not shown).

Example 3.6 Elastic Light Scattering

The tendency for aggregate formation of 23IL0075 in the differentformulation buffers was determined using elastic light scatteringmeasured at an angle of 90° by temperature-induced denaturation asmeasured in the Jasco Spectrofluorometer (excitation and emissionwavelength 500 nm). First we looked for the optimal proteinconcentration, using the 10 mM phosphate buffer pH 6.0. Atconcentrations of 175 μg/mL 23IL0075 or lower no increase in scatterintensity was seen in the tested temperature interval: (45-95° C.). Onlyat 250 μg/mL scatter was observed. The curve seemed to display twotransitions, which could indicate the formation of two different typesof aggregates (see FIG. 43).

The experiment was repeated for the acetate and the histidine buffers,both at pH 6.0. The aggregation onset temperatures in the three bufferswere very similar (Table 37). The main difference between the threebuffers was the maximum scatter: it stayed within detector range (around435 abs) for histidine while it went out of range in the phosphate aswell as in the acetate buffers (FIGS. 44 and 45). In histidine thesecond transition was absent (FIG. 46). As the scatter is proportionalto the level of aggregates formed, this indicated that the 10 mMhistidine would be a more optimal formulation buffer than 10 mM acetateand 10 mM phosphate pH 6.

Example 3.7 Freeze/Thaw and Shear Stress Study on 23IL0075 in aHistidine, Acetate and Phosphate Formulation Buffer with Mannitol or aMixture of a Mannitol and Glycine as Excipients, and a Non-IonicDetergent as Surfactant

The sensitivity of 23IL0075 to freeze/thawing and to shear or stirringhas been investigated in different candidate formulation buffers (seeTable 38). The freeze/thaw stress study consisted of 10 cycles: 100 μLsample in an eppendorf tube was frozen at −20° C. until completelyfrozen, and thawed at room temperature for 30 minutes followed by gentlemixing. The shear stress test was conducted in a cold room (4-8° C.) insmall glass tubes with 150 μl of protein solution; the protein solutionwas stirred through a magnetic bar for 4 hours at a medium rotationspeed. All samples were analyzed by RP-HPLC, SE-HPLC and OD500, somesamples also by BIACORE. On RP-HPLC, no influence of shear orfreeze/thaw was detected.

On SE-HPLC, dependent on the formulation buffer, freeze/thaw stresscaused an increase in % pre-peak up to 2.5% (FIG. 47 (B)). A mixture ofmannitol and glycine protected better against freeze/thaw stress thanonly mannitol as excipient (FIG. 47).

In the shear stressed samples hardly any increase in % pre-peak onSE-HPLC was detected but up to 10 times more opalescence (OD500) wasmeasured than in the freeze/thaw samples. Optical density at 500 nm(OD500) increased in the stirred samples and correlated with theopalescence in the samples. We conclude that in the histidine bufferwith 0.05% Poloxamer or with 0.005% TWEEN (polysorbate) 80 theopalescence remained lowest (FIG. 48).

Example 3.8 Freeze/Thaw, Shear Stress and Temperature Stress (37° C.)Stability Study for 23IL0075 in a Histidine Formulation Buffer withDifferent Combinations of Excipients

Based on the conclusions of Example 3.7, we further tested freeze/thaw,shear stress and temperature stress (37° C.) stability in differenthistidine formulation buffers (FIG. 49 and FIG. 50). Differentexcipients were tested in a formulation with 25 mg/mL of protein. Anoverview of the formulation buffers tested in F/T and storage stabilityis presented in Table 39. In FIG. 49 the results of OD500 measurementsand SE-HPLC after freeze/thaw stress are presented. A negligibleincrease of OD500 was observed. In SE-HPLC, we saw an increase ofoligomers only for the sample with 5.4% mannitol as excipient. Table 40presents the tested buffers for shear stress. Here no detergents wereincluded, to mimic the situation during the final concentration step ofthe DSP process. In FIG. 50 the OD500, SE-HPLC and BIACORE resultsobtained after 4 hours of stirring of the formulation are presented.Stirring of the sample caused increase in OD500 absorption, but noinfluence on the % soluble oligomers as measured by SE-HPLC. Thesesamples were also tested for albumin binding on BIACORE. We concludethat the protein was best protected against the shear stress by 10%sucrose, followed by a mixture of mannitol and glycine as excipients. Bycomparison of the values with the values of Example 3.7, we see that theresults were reproducible, and that for the shear stress the addition ofsome detergent was beneficial.

The accelerated stability samples at 25 mg/mL in the different candidateformulation buffers were analyzed by OD500, SE-HPLC and RP-HPLC after 3and 6 weeks storage. In SE-HPLC, the increase of oligomers was only seenat 37° C. (FIG. 51). In RP-HPLC the post-peak corresponding to thepyroglutamate increased from 3% to 4% after 6 weeks storage at 25° C.,but to on average 9% after 6 weeks at 37° C. In Table 41 the RP-HPLC andSE-HPLC results after 3 and 6 weeks storage at 37° C. and 6 weeks at 25°C. are shown. The OD500 values remained for all buffers (except oneoutlier) below 0.01.

Tables

TABLE 1 Overview of the different formulation buffers of RANKL008a usedin stability testing. Concen- Man- tration nitol RANKL008a [NaCl] %Buffer (mg/mL) Buffer (mM) (w:v) 1 60 10 mM NaH₂PO₄•2H₂O, pH 7 50 0 2 6010 mM NaH₂PO₄•2H₂O, pH 7 100 0 3 60 10 mM NaH₂PO₄•2H₂O, pH 7 0 10 4 5910 mM Na-acetate, pH 5.5 50 0 5 59 10 mM Na-acetate, pH 5.5 100 0 6 5910 mM Na-acetate, pH 5.5 0 10 7 60 20 mM L-histidine, pH 5.5 50 0 8 6020 mM L-histidine, pH 5.5 100 0 9 60 20 mM L-histidine, pH 5.5 0 10 1058 20 mM L-histidine, pH 6 50 0 11 58 20 mM L-histidine, pH 6 100 0 1258 20 mM L-histidine, pH 6 0 10 13 84.3 10 mM NaH₂PO₄•2H₂O, pH 7 100 014 70 10 mM NaH₂PO₄•2H₂O, pH 7 0 5

TABLE 2 Relative potencies of HSA and RANKL binding moieties ofRANKL008a after 10 F/T cycles as determined by the ELISA potency assays(inhibition and HSA binding). Relative potency (relative to referencematerial) Buffer RANKL HSA Phosphate + 50 mM NaCl, pH 7 0.904 0.767Phosphate + 100 mM NaCl, pH 7 0.966 0.672 Phosphate + 10% Mannitol, pH 70.956 0.715 Acetate + 50 mM NaCl, pH 5.5 1.033 0.747 Acetate + 100 mMNaCl, pH 5.5 0.905 0.705 Acetate + 10% Mannitol, pH 5.5 0.878 0.737Histidine + 50 mM NaCl, pH 5.5 0.724 0.723 Histidine + 100 mM NaCl, pH5.5 0.719 0.670 Histidine + 10% Mannitol, pH 5.5 0.692 0.572 Histidine +50 mM NaCl, pH 6 0.927 0.768 Histidine + 100 mM NaCl, pH 6 0.923 0.680Histidine + 10% Mannitol, pH 6 0.882 0.754

TABLE 3 Integration data (% of total surface area) of the differentpeaks observed in the SE-HPLC chromatograms of RANKL008a after 10 F/Tcycles or stored at 37° C. in different formulation buffers at all timepoints tested and in comparison with each control sample (each buffer).Phosphate pH 7 Phosphate pH 7 Phosphate pH 7 Acetate pH 5.5 50 mM NaCl100 mM NaCl 10% Mannitol Acetate pH 5.5 Acetate pH 5.5 10% MannitolHistidine pH 5.5 SE-HPLC Sample 60 mg/ml 60 mg/ml 60 mg/ml 50 mM NaCl100 mM NaCl 59 mg/ml 50 mM NaCl % control 0 0 0 0 0 0 0 Prepeak 10 F/Tcycles 0 0 0 0 0 0 0 2 w 37° C. 5.6 6.9 1.3 4.6 6.3 2.3 5.5 3 w 37° C.4.4 6.2 0.65 3.9 5.9 0.18 5.6 5 w 37° C. 13.7 15.8 3.9 11.5 14.2 1.2214.0 10 w 37° C.  23.8 25.3 11.1 21.0 23.9 3.4 27.2 % Main control 100100 100 100 100 100 100 peak 10 F/T cycles 100 100 100 100 100 100 100 2w 37° C. 93.5 92.2 97.9 94.8 93.1 98.8 94.0 3 w 37° C. 93.7 92.0 95.295.0 92.8 96.9 93.4 5 w 37° C. 81.14 78.87 91.52 87.38 84.63 97.87 84.8510 w 37° C.  69.2 68.0 80.5 77.5 74.7 95.1 71.3 % control 0 0 0 0 0 0 0Postpeak1 10 F/T cycles 0 0 0 0 0 0 0 2 w 37° C. 0 0 0 0 0 0 0 3 w 37°C. 0 0 0 0 0 0 0 5 w 37° C. 3.16 3.36 3.12 0 0 0 0 10 w 37° C.  3.7 3.55.0 0 0 0 0 % control 0 0 0 0 0 0 0 Postpeak2 10 F/T cycles 0 0 0 0 0 00 2 w 37° C. 0.23 0.27 0.19 0.23 0.26 0.19 0.19 3 w 37° C. 0.57 0.580.31 0.49 0.53 0.27 0.48 5 w 37° C. 0.41 0.47 0.27 0.37 0.39 0.25 0.4510 w 37° C.  0.5 0.5 0.3 0.4 0.4 0.2 0.4 % control 0 0 0 0 0 0 0Postpeak3 10 F/T cycles 0 0 0 0 0 0 0 2 w 37° C. 0.62 0.64 0.60 0.370.41 0.46 0.31 3 w 37° C. 1.15 1.25 1.07 0.52 0.64 0.61 0.49 5 w 37° C.1.59 1.50 1.49 0.75 0.78 0.66 0.70 10 w 37° C.  2.7 2.6 3.1 1.1 1.0 1.31.1 Histidine pH 5.5 Histidine pH 6 Histidine pH 5.5 10% MannitolHistidine pH 6 Histidine pH 6 10% Mannitol SE-HPLC Sample 100 mM NaCl 60mg/ml 50 mM NaCl 100 mM NaCl 58 mg/ml % control 0 0   0 0 0   Prepeak 10F/T cycles 0 0   0 0 0   2 w 37° C. 7.5 0.54 6.3 7.7 0.63 3 w 37° C. 7.90.34 7.0 8.6 0.39 5 w 37° C. 17.1 1.5  16.2 17.4 2.0  10 w 37° C.  27.85.4  26.8 27.0 7.3  % Main control 100 100*    100 100 100*    peak 10F/T cycles 100 100    100 100 100    2 w 37° C. 92.1 98.8  93.1 91.596.7  3 w 37° C. 91.5 98.6  91.3 90.2 98.8  5 w 37° C. 81.73 97.49 82.22 81.19 96.76  10 w 37° C.  73.5 93.1  71.3 71.2 91.0  % control 00   0 0 0   Postpeak1 10 F/T cycles 0 0   0 0 0   2 w 37° C. 0 0   0 00   3 w 37° C. 0 0   0 0 0   5 w 37° C. 0 0   0 0 0   10 w 37° C.  0 0  0 0 0   % control 0 0   0 0 0   Postpeak2 10 F/T cycles 0 0   0 0 0   2w 37° C. 0.17 0.19 0.20 0.23 0.18 3 w 37° C. 0.55 0.27 0.54 0.5 0.27 5 w37° C. 0.29 0.23 0.52 0.42 0.37 10 w 37° C.  0.5 0.2  0.4 0.4 0.3  %control 0 0   0 0 0   Postpeak3 10 F/T cycles 0 0   0 0 0   2 w 37° C.0.26 0.37 0.40 0.58 0.53 3 w 37° C. 0.55 0.57 1.12 0.71 0.56 5 w 37° C.0.88 0.78 1.06 0.99 0.87 10 w 37° C.  1.3 1.3  1.5 1.4 1.5 

TABLE 4 Integration data (% of total surface area) of the differentpeaks observed in the RP-HPLC chromatograms of RANKL008a after 10 F/Tcycles or stored at 37° C. in different formulation buffers at all timepoints tested and in comparison with each control sample (each buffer)Phosphate pH 7 Phosphate pH 7 Phosphate pH 7 Acetate pH 5.5 50 mM NaCl100 mM NaCl 10% Mannitol Acetate pH 5.5 Acetate pH 5.5 10% MannitolHistidine pH 5.5 RP-HPLC Sample 60 mg/ml 60 mg/ml 60 mg/ml 50 mM NaCl100 mM NaCl 59 mg/ml 50 mM NaCl % control 0.25 0.14 0 0 0 ND 0 Prepeak 110 F/T cycles 0.20 0.08 0 0 0 0 0 2 w 37° C. 0 0 0 0 0 0 0 3 w 37° C. 00 0 0 0 0 0 5 w 37° C. 0 0 0 0 0 0 0 10 w 37° C.  0 0 0 0 0 0 0 %Control 0 0 0 0 0 ND 0 Prepeak 2 10 F/T cycles 0 0 0 0 0 0 0 2 w 37° C.1.10 1.00 1.10 0.78 0.82 0.78 0.82 3 w 37° C. 1.6 1.4 1.8 0.7 0.8 0.90.7 5 w 37° C. 2.1 2.0 2.3 1.0 0.9 1.1 1.1 10 w 37° C.  3.4 2.9 3.7 1.51.5 1.9 1.6 % Main Control 96.6 96.7 96.7 96.8 97.0 ND 96.5 peak 10 F/Tcycles 96.7 96.6 95.9 97.0 96.7 96.6 97.0 2 w 37° C. 93.7 94.0 93.5 95.896.0 95.6 96.0 3 w 37° C. 92.3 92.7 91.9 95.7 95.5 95.4 95.6 5 w 37° C.90.2 90.3 89.1 94.8 95.1 94.7 94.7 10 w 37° C.  84.1 85.4 83.4 93.5 93.392.7 93.3 % Control 3.2 3.1 3.3 3.2 2.9 ND 3.5 Pyroglut. 10 F/T cycles3.1 3.3 4.0 3.3 3.2 3.4 3.0 2 w 37° C. 5.1 4.7 5.4 3.4 3.2 3.6 3.2 3 w37° C. 6.1 6.0 6.3 3.6 3.7 3.7 3.7 5 w 37° C. 7.7 7.7 8.6 4.1 3.9 4.24.2 10 w 37° C.  12.5 11.7 12.9 5.0 5.2 5.5 5.2 Histidine pH 5.5Histidine pH 6 Histidine pH 5.5 10% Mannitol Histidine pH 6 Histidine pH6 10% Mannitol RP-HPLC Sample 100 mM NaCl 60 mg/ml 50 mM NaCl 100 mMNaCl 58 mg/ml % control 0 0 0 0 0 Prepeak 1 10 F/T cycles 0 0 0 0 0 2 w37° C. 0 0 0 0 0 3 w 37° C. 0 0 0 0 0 5 w 37° C. 0 0 0 0 0 10 w 37° C. 0 0 0 0 0 % Control 0 0 0 0 0 Prepeak 2 10 F/T cycles 0 0 0 0 0 2 w 37°C. 0.72 0.76 0.76 0.79 0.87 3 w 37° C. 0.9 0.9 0.7 0.9 0.9 5 w 37° C.1.2 1.2 1.2 1.1 1.5 10 w 37° C.  1.7 2.0 2.0 1.9 2.1 % Main Control 96.696.4 96.7 96.8 96.8 peak 10 F/T cycles 95.8 96.5 96.4 96.8 96.8 2 w 37°C. 95.7 95.9 95.6 95.7 95.5 3 w 37° C. 95.4 95.3 95.6 95.5 95.5 5 w 37°C. 94.6 94.8 94.4 94.7 94.0 10 w 37° C.  93.3 92.4 93.1 92.3 92.0 %Control 3.0 3.6 3.3 3.2 3.2 Pyroglut. 10 F/T cycles 3.3 3.5 3.6 3.2 3.22 w 37° C. 3.5 3.4 3.6 3.5 3.6 3 w 37° C. 3.7 3.7 3.6 3.6 3.6 5 w 37° C.4.3 4.1 4.4 4.1 4.4 10 w 37° C.  5.1 5.5 4.8 5.8 5.9

TABLE 5 Integration data (% of total surface area) of the differentpeaks observed in the IEX-HPLC chromatograms of RANKL008a stored for 10weeks at 37° C. in different formulation buffers. % Main % Post % PostBuffer peak peak 1 peak 2 Phosphate + 50 mM NaCl, pH 7 69.6 4.4 26.0Phosphate + 100 mM NaCl, pH 7 67.0 4.5 28.1 Phosphate + 10% Mannitol, pH7 84.6 3.2 12.1 Acetate + 50 mM NaCl, pH 5.5 72.8 2.7 24.4 Acetate + 100mM NaCl, pH 5.5 69.6 2.8 27.6 Acetate + 10% Mannitol, pH 5.5 95.4 0 4.6Histidine + 50 mM NaCl, pH 5.5 66.0 2.8 31.2 Histidine + 100 mM NaCl, pH5.5 68.1 2.8 29.0 Histidine + 10% Mannitol, pH 5.5 92.8 0 7.2Histidine + 50 mM NaCl, pH 6 67.4 2.6 30.1 Histidine + 100 mM NaCl, pH 667.0 2.6 30.4 Histidine + 10% Mannitol, pH 6 88.8 2.6 9.0

TABLE 6 Relative potencies of the HSA and RANKL binding moieties ofRANKL008a after 10 weeks at 37° C. as measured by BIACORE analysis.Relative potency Buffer RANKL HSA Phosphate + 50 mM NaCl, pH 7 81.0 57.4Phosphate + 100 mM NaCl, pH 7 78.6 56.6 Phosphate + 10% Mannitol, pH 776.3 66.8 Acetate + 50 mM NaCl, pH 5.5 80.1 63.0 Acetate + 100 mM NaCl,pH 5.5 78.0 59.0 Acetate + 10% Mannitol, pH 5.5 80.9 79.4 Histidine + 50mM NaCl, pH 5.5 80.2 59.7 Histidine + 100 mM NaCl, pH 5.5 73.1 55.0Histidine + 10% Mannitol, pH 5.5 75.2 73.6 Histidine + 50 mM NaCl, pH 679.1 59.3 Histidine + 100 mM NaCl, pH 6 78.3 57.5 Histidine + 10%Mannitol, pH 6 87.4 83.4

TABLE 7 Visual inspection and content determination of RANKL008a afterdilution to 0.28 mg/mL in different diluents and passage/storage insyringes (refer to FIG. 9). content (mg/mL) (95% Sample* visualinspection confidence interval 0028 SALINE TUB small precipitates 0.265(0.261-0.270) 0028 SALINE SYR S25/0 small precipitates 0.263(0.261-0.265) 0028 SALINE SYR S25/24 small precipitates 0.259(0.256-0.262) 0028 PLACEBO − TW TUB small precipitates 0.272(0.271-0.273) 0028 PLACEBO − TW SYR small precipitates 0.268(0.267-0.269) S25/0 0028 PLACEBO − TW SYR small precipitates 0.268(0.259-0.276) S25/24 0028 PLACEBO + TW TUB clear 0.281 (0.281-0.281)0028 PLACEBO + TW SYR slightly turbid 0.280 (0.278-0.282) S25/0 0028PLACEBO + TW SYR slightly turbid 0.279 (0.277-0.281) S25/24 *S25/0:storage at 25° C. for 0 minute S25/24: storage at 25° C. for 24 h −TW:buffer minus TWEEN (polysorbate) 80 +TW: buffer + TWEEN (polysorbate) 80PLACEBO refers to the following buffer: 10 mM Na2HPO4 pH 7.0 + 115 mMNaCl TUB: sample stored in a polystyrene tube

TABLE 8 Visual inspection, content (confidence interval) and turbidityof RANKL008a diluted to 0.28 mg/mL before (TUB) and after passagethrough syringes with different needle size as described in Example 1.8.visual content (mg mL) OD 320/278 OD 350/278 Sample* inspection (95%confidence interval) ratio ratio 0028 PLACEBO + TW TUB clear 0.288(0.275-0.301) 0.0010 0.0019 0028 PLACEBO + TW 18G/18G clear 0.285(0.284-0.286) 0.0003 0.0000 0028 PLACEBO + TW 18G/23G clear 0.288(0.271-0.307) 0.0000 0.0000 0028 PLACEBO + TW 18G/27G clear 0.285(0.279-0.290) 0.0000 0.0002 0028 PLACEBO + TW 18G/30G clear 0.286(0.285-0.287) 0.0005 0.0002 0028 PLACEBO + TW 23G/23G clear 0.287(0.285-0.289) 0.0005 0.0007 0028 PLACEBO + TW 27G/27G clear 0.285(0.284-0.286) 0.0001 0.0005 0028 PLACEBO + TW 30G/30G clear 0.287(0.280-0.294) 0.0007 0.0019 *+TW: buffer + TWEEN (polysorbate) 80PLACEBO refers to the following buffer: 10 mM Na2HPO4 pH 7.0 + 115 mMNaCl TUB: sample stored in a polystyrene tube 18G/18G: sample drawn upwith a 18G needle and expelled through a 18G needle 18G/27G: drawn upwith a 18G needle and expelled through a 27G needleAll other coding is similar to the two examples given above

TABLE 9 Visual inspection, content (with 95% confidence interval) andturbidity of RANKL008a before (TUB) and after passage through syringeswith different needle size as described in Example 1.7 at aconcentration of 0.28 mg/mL or about 65 mg/mL. visual content (mg/mL) OD320/278 OD 350/278 Sample* inspection (95% confidence interval) ratioratio 0028 PLACEBO + TW TUB clear 0.284 (0.283-0.285) 0.0014 0.0010 0028PLACEBO + TW 27G/27G (3x) clear 0.284 (0.283-0.285) 0.0031 0.0021 0028PLACEBO + TW 29G/29G B clear 0.282 (0.280-0.284) 0.0024 0.0010 0028PLACEBO + TW 29G/29G T clear 0.283 (0.282-0.284) 0.0041 0.0033 6500PLACEBO + TW TUB clear 63.5 (62.4-64.6)  0.0019 0.0006 6500 PLACEBO + TW27G/27G (3x) clear 62.9 (62.7-63.1)  0.0015 0.0008 *+TW: buffer + TWEEN(polysorbate) 80 PLACEBO refers to the foiiowing buffer: 10 mM Na2HPO4pH 7.0 + 115 mM NaCl TUB: sample stored in a polystyrene tube 27G/27G:sample drawn up with a 27G needle and expelled through a 27G needle29G/29G: drawn up with a 29G needle and expelled through a 29G needle T:Terumo needle, B Becton Dickinson needle 0028 refers to concentration at0.28 mg/mL, 6500 to 65 mg/mL

TABLE 10 Protein sequences of Nanobodies used in Example 2IL6R304, SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTTSRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLOMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS IL6R305, SEQ ID NO: 2EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFNINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS IL6R306, SEQ ID NO: 3EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFNINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS

TABLE 11 Overview of the IL6R batches used in the formulation armstability studies described in Example 2. Batch no. Nanobody BufferConcentration P#051108nr1 IL6R304 PBS 4.58 mg/mL P#051108nr2 IL6R305 PBS3.46 mg/mL P#051108nr2 IL6R306 PBS 5.65 mg/mL B5#030309nr1.5-9 IL6R30425 mM hepes, pH 75, 3.79 mg/mL 100 mM NaCl B5#060509nr1 IL6R304 25 mMhepes, pH 7.5,  4.6 mg/mL 100 mM NaCl

TABLE 12 Reagents used in the formulation and stability study describedin Example 2 Reagent Provider Cat No. ACN, HPLC grade Biosolve Cat. No.012007 TFA Biosolve Cat. No. 20234131 Isopropanol, HPLC grade BiosolveCat. No. 162606 MILLI-Q grade water D-PBS Gibco Cat. No. 14190-094 NaClMerck Cat. No. 1.06404.1000 Gel filtration standard Bio-Rad Cat. No.151-1901 HSA Sigma Cat. No. A3782 L-histidine Fluka Cat. No. 53319D-Mannitol Fluka Cat. No. 17311 Sucrose Fluka Cat. No. 18219 GlycineFluka Cat. No. 50058 Tween-80 Merck Cat. No. K351 65661 609L-histidine-HCl monohydrate Sigma Cat. No. 53369 Succinic acid disodiumFluka Cat. No. 14158 hexahydrate TRIZMA base Sigma Cat. No. T6066-5Sorbitol Fluka Cat. No. 85529 Xylitol Sigma Cat. No. X3375-100g RibitolFluka Cat. No. 02240 L-Arginine Fluka Cat. No. 11009 MES Sigma Cat. No.M3671 Sodium dihydrogenphosphate Merck Cat. No. 1.06345.1000 Disodiumhydrogenphosphate Merck Cat. No. 1.06576.1000

TABLE 13 Summary of the results from the TSA assay.

The Tm values obtained in the different buffer are coded from white todark grey, i.e. from higher to lower Tm values.

TABLE 14 Overview of the Tm values obtained by DSC using IL6R304. Tm (°C.) Tm (° C.) Tm (° C.) Tm (° C.) in in in 0 mM in 25 mM 100 mM 500 mMBuffer NaCl NaCl NaCl NaCl 25 mM citrate pH 3.5 50.21 / / / 25 mMacetate pH 5.5 61.30 61.21 60.19 58.59 25 mM MES pH 6.0 62.52 61.8360.60 58.66 25 mM hepes pH 7.0 62.48 62.27 61.13 59.24 25 mM phosphatepH 7.0 60.70 / / / 25 mM Tris pH 7.5 61.82 61.82 60.91 59.43

TABLE 15 Overview of the Tm results obtained by DSC and TSA usingIL6R304 formulated in different buffers. DSC Tm (° C.) TSA in 15 mM Tm(° C.) Tm (° C.) Tm (° C.) L- 15 mM in 15 mM 15 mM histidine, phosphate,L-histidine, phosphate, Excipient(s) pH 6.5 pH 6.5 pH 6.5 pH 6.5 / ND ND 61.09*/61.11** 60.48/60.48 5% mannitol 64.74 63.81 61.91/61.8761.34/61.31 10% sucrose 65.40 64.35 62.78/63.20 62.34/62.78 10%trehalose 65.28 64.51 62.78/63.59 63.17/63.61 2.5% 64.37 63.8361.52/61.91 61.53/61.30 mannitol + 2.5% sucrose 2.5% sorbitol + 64.6563.85 62.34/62.34 62.77/62.77 2.5% trehalose 2.5% sorbitol + 64.83 64.3362.33/62.35 63.19/62.96 2.5% trehalose + 1.5 mM Glycine 10% Run 63.77 NDND trehalose + failure 0.01% Tween- 80 *measurement 1, **measurement 2

TABLE 16 Visual appearance, UV spectroscopy and PAMAS data demonstratinga higher solubility for IL6R304 in the presence of TWEEN (polysorbate)80. IL6R304 (2 mg/mL) stored for 4 days at 5° C. PBS + PBS + 0.1% 0.2%TWEEN PBS TWEEN 80 80 Appearance Turbid, Clear, colorless Clear,colorless opaque A320/A280 0.014 0.012 0.009 Sub-visible particlecounts/ 100 μl  >1 μm 157.557 42.243 52.157  >2 μm 69.471 19.514 21.429 >3 μm 43.371 12.043 12.743  >4 μm 29.757 8.329 8.600  >5 μm 18.3004.971 4.800  >6 μm 11.814 3.114 3.143  >7 μm 8.300 2.300 2.186  >8 μm5.900 1.671 1.500  >9 μm 4.543 1.214 1.086  >10 μm 3.400 971 800  >15 μm800 271 300  >25 μm 200 100 86  >50 μm 114 29 14 >100 μm 86 0 14 >150 μm71 0 14 >200 μm 71 0 14

TABLE 17 Overview of the different formulation buffers used in initialstability testing of IL6R304, IL6R305 and IL6R306. Condition Buffer[NaCl] Mannitol 1 PBS  0 mM 0% 2 PBS  0 mM 5% 3 10 mM NaH₂PO₄•2H₂O, pH 7100 mM 0% 4 10 mM NaH₂PO₄•2H₂O, pH 7 100 mM 5% 5 10 mM Na-acetate, pH5.5 100 mM 0% 6 10 mM Na-acetate, pH 5.5 100 mM 5% 7 20 mM L-histidine,pH 6 100 mM 0% 8 20 mM L-histidine, pH 6 100 mM 5%

TABLE 18 Overview of the different formulation buffers used in stabilitytesting of IL6R304. Concen- % % % mM Buff- tration TWEEN Man- Su- Gly-er IL6R304 Buffer 80 nitol crose cine 1 10 mg/mL 20 mM L- / / / /histidine 2 10 mg/mL 20 mM L- 0.01 / / / histidine 3 10 mg/mL 20 mM L-0.05 / / / histidine 4 10 mg/mL 20 mM L- 0.05 5 / / histidine 5 10 mg/mL20 mM L- 0.05 5 / 200 histidine 6 10 mg/mL 20 mM L- 0.05 2.5 / 100histidine 7 10 mg/mL 20 mM L- 0.05 / 10  / histidine 8 10 mg/mL 20 mM L-0.05 / / 200 histidine 9 10 mg/mL 20 mM L- 0.05 / 5 100 histidine 10 10mg/mL 20 mM L- 0.05 2.5 5 / histidine 11 10 mg/mL 20 mM L- / 2.5 5 100histidine 12 10 mg/mL 20 mM L- 0.05 2.5 5 100 histidine

TABLE 19 Methods used for assessing the stability of IL6R304 atdifferent time points (represented as x weeks or w) after storage at 5°C. and 37° C. Ref. Stress condition Method Purpose material 5° C. 37° C.A280 Content 0 w 1, 2 and 5 w 1, 2, 3 and 5 w Appearance Precipitation 0w 1, 2 and 5 w 1, 2, 3 and 5 w RP-HPLC Purity/variants 0 w 1, 2 and 5 w1, 2, 3 and 5 w SE-HPLC Purity/ 0 w 1, 2 and 5 w 1, 2, 3 and 5 waggregation/ hydrolysis BIACORE Potency 0 w 5 w 5 w (HSA binding)Osmolality Characteristic 0 w / /

TABLE 20 Overview of the SE-HPLC integration results after storage for 6months at 37° C. Buffer % pre peak 1 % pre peak 2 % main peak % postpeak Ref 0.52 0.17 99.3 0 Buffer 1 ND ND ND ND Buffer 2 20.4 2.1 73.44.1 Buffer 3 ND ND ND ND Buffer 4 18.1 1.7 76.0 4.2 Buffer 5 22.2 2.071.4 4.4 Buffer 6 21.4 1.7 72.7 4.2 Buffer 7 15.1 0 80.5 4.4 Buffer 821.1 2.4 72.0 4.5 Buffer 9 16.7 2.7 76.3 4.3 Buffer 10 15.8 1.9 77.9 4.4Buffer 11 17.5 2.0 76.4 4.2 Buffer 12 16.8 3.3 75.7 4.2

TABLE 21 Overview of the different formulation buffers tested in thestability study. Nr. Conc. Buffer Mannitol Sucrose Trehalose GlycineTWEEN-80 1 10 mg/mL 15 mM L-histidine, pH 6.5  5% 0.01% 2 10 mg/mL 15 mML-histidine, pH 6.5 10% 0.01% 3 10 mg/mL 15 mM L-histidine, pH 6.5 10%0.01% 4 10 mg/mL 15 mM L-histidine, pH 6.5 7.5%  0.35% 0.01% 5 10 mg/mL15 mM L-histidine, pH 6.5 2.5%  5% 0.01% 6 10 mg/mL 15 mM phosphate, pH6.5  5% 0.01% 7 10 mg/mL 15 mM phosphate, pH 6.5 10% 0.01% 8 10 mg/mL 15mM phosphate, pH 6.5 10% 0.01% 9 10 mg/mL 15 mM phosphate, pH 6.5 7.5% 0.35% 0.01% 10 10 mg/mL 15 mM phosphate, pH 6.5 2.5%  5% 0.01%

TABLE 22 cIEF integration data of IL6R304 stored for 8 weeks at 37° C.in the different buffers % prepeak % postpeak Buffer (acidic variants) %main peak (basic variants) 1 5.5 81.3 13.0 2 5.0 81.5 13.5 3 6.1 79.714.2 4 5.7 81.2 13.2 5 5.1 81.2 13.7 6 9.0 71.6 19.3 7 9.9 70.5 19.6 88.3 71.8 19.9 9 11.7 68.5 19.8 10 8.7 70.5 20.2

TABLE 23 Relative potency of IL6R304 after 8 weeks at +37° C. comparedto B5#030309nr2.3-5. Buffer HSA IL-6R 1 1.080 (0.954-1.223) 1.153(0.957-1.389) 2 0.975 (0.887-1.072) 0.980 (0.760-1.263) 3 1.038(0.952-1.132) 1.117 (0.910-1.372) 4 1.182 (1.074-1.300) 1.061(0.908-1.240) 5 1.080 (1.004-1.161) 1.082 (0.925-1.266)

TABLE 24 Summary of the BIACORE results for HSA binding of the stabilitysamples stored for 8 weeks at 37° C., expressed as % activity comparedto the equivalent sample stored at −70° C. Buffer % activity compared toreference 1 97.5 2 93.2 3 92.5 4 83.9 5 101.9 6 92.2 7 89.4 8 99.0 984.3 10 89.6

TABLE 25 Appearance of IL6R304 after 0, 2, 4 and 24 hours of stirring at2-8° C. Buffer 0 hrs 2 hrs 4 hrs 24 hrs 1 clear clear clearclear/slightly opalescent 2 clear clear clear clear/slightly opalescent3 clear clear slightly opalescent slightly opalescent 4 clear clearclear clear/slightly opalescent 5 clear clear slightly opalescentslightly opalescent 6 clear clear slightly opalescent opalescent 7 clearclear slightly opalescent opalescent 8 clear clear slightly opalescenthighly opalescent 9 clear clear clear opalescent 10 clear clearopalescent opalescent

TABLE 26 Reagents used in the formulation and stability study describedin Examples 3. Reagent Provider Cat No. ACN, HPLC grade Biosolve Cat.No. 012007 TFA Biosolve Cat. No. 20234131 N-propanol, HPLC gradeSigma-Aldrich Cat. No. 34871 MILLI-Q grade water D-PBS Invitrogen Cat.No. 14190 NaCl Merck Cat. No. 1.06404.1000 Gel filtration standardBio-Rad Cat. No. 151-1901 HSA Sigma Cat. No. A3782 L-histidine FlukaCat. No. 53319 D-Mannitol Fluka Cat. No. 17311 Sucrose Fluka Cat. No.18219 Glycine Fluka Cat. No. 50058 TWEEN 80 Merck Cat. No. K351 65661609

TABLE 27 Final concentrations obtained after concentration of 23IL0064using VIVASPIN filters. The filtration was stopped at the moment thefinal volume became limited (100 to 200 μL) and protein loss occurred.All samples were analyzed by SE-HPLC: the percent pre-peak (% of totalsurface area) represent aggregates in the sample. start conditions afterULTRAFILTRATION % pre-peak % pre-peak buffer condition concentration inSE-HPLC concentration in SE-HPLC % recovery D-PBS 3.8 mg/mL 2.80% 110mg/mL 6.30% 42%⁽¹⁾ 50 mM NaCl, 3.4 mg/mL 2.70%  83 mg/mL 2.80% 66%⁽¹⁾ 10mM Phosphate pH 7 50 mM NaCl, 3.4 mg/mL 2.70% 150 mg/mL 2.70% 59%⁽¹⁾ 40mM Histidine pH 6 ⁽¹⁾Low recoveries can be due to local concentrationeffects obtained during the dead-end ultrafiltration set-up.

TABLE 28 Buffers tested in a thermal shift assay for 23IL0064.Concentration Buffer pKa pH (mM) mM NaCl % mannitol Succinate 5.64 (pK2)5.2 20 0 0-2.5-5-7.5 6.2 50-150- 0 300-500 Histidine 6.04 (pK2) 5.5 20 00-2.5-5-7.5 6.5 50-150- 0 300-500 Phosphate 7.20 (pK2) 6.7 20 00-2.5-5-7.5 7.7 50-150- 0 300-500 hepes 7.48 7.0 20 0 0-2.5-5-7.5 8.050-150- 0 300-500

TABLE 29 Concentrations measured by NANODROP (average of 2 measurements)in the samples stressed at 37° C. The concentration was determined aftera short high speed spin (1 min at 15000xg) P23IL0064 Samples (4 mg/mL inD-PBS) Concentration (mg/mL) Reference (−20° C.) 4.05 3 weeks 37° C.(label 24 w) 3.99 4 weeks 37° C. (label 4 w) 2.0 6 weeks 37° C. (label 8w) 2.30 6 weeks 37° C. (label 12 w) 4.11 6 weeks 37° C. (label 16 w)2.33

TABLE 30 Buffers tested in stressed stability for 23IL0064. Excipient/Time point Storage Buffer pH Concentration* surfactant analyzed Temp.Hepes 20 mM 8 5 mg/mL 2.5 w 3° C. His 20 mM 6.5 5 mg/mL 2.5 w/6 w4-25-37° C. His 20 mM 6.5  22 mg/mL** 2.5 w/6 w 4-25-37° C. His 20 mM 65 mg/mL 2.5 w/6 w 4-25-37° C. His 20 mM 6.5 5 mg/mL 0.02% TWEEN 80 2.5w/6 w 4-25-37° C. His 20 mM 6.5 5 mg/mL   8% mannitol 2.5 w/6 w 4-25-37°C. His 20 mM 6.5 5 mg/mL   % sucrose 2.5 w/6 w 4-25-37° C. His 20 mM 6.55 mg/mL  1.5% glycine 2.5 w/6 w 4-25-37° C. *The exact concentrationsused in the study ranged between 4.9 and 5.1 mg/mL **Labeled as ‘HIGHCONC’ in figures, actual conc. was 22.36 mg/mL.

TABLE 31 Crude ranking of the degree of opalescence and material lossinduced by shear stress for 23IL0064 in different formulation buffers.

TABLE 32 Integration data of the RP-HPLC analysis of the stabilitysamples of 23IL0064 in different buffer conditions (comparison of 6weeks 37° C., 6 weeks 25° C., 6 weeks 4° C. and −80° C. Ref). Stress pre1 pre 2 main peak post 1 post 2 Total peak p23IL0064 in condition 22.423.3 23.9 27.5 29.0 area (%)* 20 mM −80° C. Ref 68 195 1671 76 20 2029Histidine % 3% 10% 82% 4% 1% 100% pH 6.5 6 weeks 4° C. 77 201 1791 85 292182 % 4%  9% 82% 4% 1% 108% 6 weeks 25° C. 77 196 1696 125  33 2127 %4%  9% 80% 6% 2% 105% 6 weeks 37° C. 130  186 1499 274  57 2146 % 6%  9%70% 13%  3% 106% 20 mM −80° C. Ref 68 199 1807 66 20 2159 Histidine % 3% 9% 84% 3% 1% 100% pH 6.0 6 weeks 4° C. 69 195 1751 71 17 2104 % 3%  9%83% 3% 1%  97% 6 weeks 25° C. 67 206 1696 90 22 2080 % 3% 10% 82% 4% 1% 96% 6 weeks 37° C. 117  200 1573 152  37 2079 % 6% 10% 76% 7% 2%  96%20 mM −80° C. Ref 66 175 1564 49 14 1867 Histidine % 4%  9% 84% 3% 1%100% pH 6.5 + 6 weeks 4° C. 74 196 1749 85 29 2133 0.02% % 3%  9% 82% 4%1% 114% TWEEN 80 6 weeks 25° C. 86 196 1556 117  37 1992 % 4% 10% 78% 6%2% 107% 6 weeks 37° C. 116  195 1258 245  66 1881 % 6% 10% 67% 13%  4%101% 20 mM −80° C. Ref 70 201 1770 66 19 2128 Histidine % 3%  9% 83% 3%1% 100% pH 6.5 + 6 weeks 4° C. 68 193 1739 80 14 2093 8% mannitol % 3% 9% 83% 4% 1%  98% 6 weeks 25° C. 82 192 1665 123  32 2094 % 4%  9% 80%6% 2%  98% 6 weeks 37° C. 95 173 1438 256  35 1998 % 5%  9% 72% 13%  2% 94% 20 mM −80° C. Ref 56 193 1685 70 16 2019 Histidine % 3% 10% 83% 3%1% 100% pH 6.5 + 6 weeks 4° C. 61 198 1691 62 17 2029 8% sucrose % 3%10% 83% 3% 1% 100% 6 weeks 25° C. 80 202 1591 123  29 2026 % 4% 10% 79%6% 1% 100% 6 weeks 37° C. 139  246 1292 316  73 2065 % 7% 12% 63% 15% 4% 102% 20 mM −80° C. Ref 53 167 1413 51 15 1698 Histidine % 3% 10% 83%3% 1% 100% pH 6.5 + 6 weeks 4° C. 68 197 1669 67 18 2019 1.5% glycine %3% 10% 83% 3% 1% 119% 6 weeks 25° C. 74 189 1596 124  31 2014 % 4%  9%79% 6% 2% 119% 6 weeks 37° C. 95 185 1359 278  52 1968 % 5%  9% 69% 14% 3% 116% 20 mM −80° C. Ref 72 233 1984 81 15 2385 Histidine % 3% 10% 83%3% 1% 100% pH 6.5 6 weeks 4° C. 90 218 1880 88 21 2297 CONC % 4%  9% 82%4% 1%  96% 6 weeks 25° C. 96 235 1848 143  33 2355 % 4% 10% 78% 6% 1% 99% 6 weeks 37° C. 126  261 1725 332  62 2505 % 5% 10% 69% 13%  2% 105%(%)* recovery calculated by using the total area compared to the −80° C.Ref of the same condition.

TABLE 33 Melting temperatures for 23IL0064 and 23IL0075 in differentbuffers as determined by differential scanning calorimetry (at 1 mg/mL).Scanning was performed at 1° C./min, starting at 30° C. Buffer 23IL006423IL0075 25 mM acetate; pH 5.5; 50 mM NaCl 55.5 58.0 25 mM acetate; pH5.5; 250 mM NaCl 52.9 55.5 25 mM MES; pH 6.0; 50 mM NaCl 56.2 58.8 25 mMMES; pH 6.0; 250 mM NaCl 53.2 55.7 25 mM hepes; pH 7.0; 50 mM NaCl 56.759.3 25 mM hepes; pH 7.0; 250 mM NaCl 53.7 56.2 25 mM Tris; pH 7.5; 50mM NaCl 56.3 58.7 25 mM Tris; pH 7.5; 250 mM NaCl 53.5 56.1

TABLE 34 Melting temperatures for 23IL0064 and 23IL0075 in buffers asdetermined by thermal shift assay (at 0.1 mg/mL). Buffer 23IL006423IL0075* 23IL0075* 20 mM Histidine pH 6.5; 54.5 57.0 57.0 50 mM NaCl 20mM Histidine pH 6.5 56.6 59.2 59.3 20 mM Histidine pH 6.5; 57.9 60.760.6 7 5% mannitol 20 mM hepes pH 7; 50 mM 54.8 57.4 57.5 NaCl 20 mMhepes pH 7 56.8 59.8 59.9 20 mM hepes pH 7; 7.5% 58.2 61.1 61.2 mannitol20 mM hepes pH 8; 50 mM 55.0 57.6 57.8 NaCl 20 mM hepes pH 8 56.4 59.559.4 20 mM hepes pH 8; 7.5% 57.6 60.3 60.3 mannitol *Measurements wereperformed on the 2 batches

TABLE 35 Design-Expert Numerical Optimization of the model. The largerthe Desirability coefficient the better the proposal of the optimum.Conc(log10) Tm Number (mM) PH Buffer (° C.) Desirability Solutions forphosphate 1.00 6.00 Phosphate 59.0012 0.895 2 1.04 6.00 Phosphate58.9419 0.881 Solutions for Acetate 1 1.00 6.00 Acetate 59.2759 0.959 21.01 6.08 Acetate 59.2615 0.956 3 1.11 5.88 Acetate 59.1489 0.929 4 1.105.80 Acetate 59.1345 0.926 5 1.15 5.72 Acetate 59.0452 0.905 6 1.16 5.68Acetate 59.0036 0.895 7 1.32 6.09 Acetate 58.9476 0.882 8 1.33 6.15Acetate 58.9406 0.881 9 1.35 6.21 Acetate 58.9317 0.879 10 1.19 5.56Acetate 58.8937 0.870 11 1.32 5.89 Acetate 58.8726 0.865 12 1.00 5.00Acetate 58.77 0.841 13 1.35 5.69 Acetate 58.7182 0.829 14 1.50 6.12Acetate 58.7181 0.829 15 1.10 5.10 Acetate 58.682 0.820 16 1.16 5.19Acetate 58.6346 0.809 17 1.57 6.08 Acetate 58.5857 0.798 18 1.44 5.71Acetate 58.5814 0.797 19 1.35 5.50 Acetate 58.5692 0.794 20 1.55 5.96Acetate 58.5672 0.793 21 1.6 6.18 Acetate 58.5354 0.786 22 1.70 6.00Acetate 58.3294 0.738 23 1.62 5.68 Acetate 58.215 0.711 24 1.34 5.01Acetate 58.0486 0.672 25 1.47 5.20 Acetate 57.9981 0.660 26 1.33 4.96Acetate 57.9943 0.659 27 1.56 5.35 Acetate 57.9714 0.654 28 1.27 4.83Acetate 57.9579 0.651 29 1.62 5.41 Acetate 57.9098 0.639 30 1.53 5.16Acetate 57.8061 0.615 31 1.61 5.29 Acetate 57.7715 0.607 32 1.57 5.20Acetate 57.7583 0.604 33 1.56 5.18 Acetate 57.7496 0.602 34 1.35 4.79Acetate 57.7035 0.591 35 1.65 5.24 Acetate 57.61 0.569 36 1.65 5.17Acetate 57.5141 0.547 37 1.70 5.00 Acetate 57.0985 0.449 37 Solutionsfound Solutions for Histidine 1 1.01 6.19 Histidine 59.4631 1.000 2 1.006.18 Histidine 59.4654 1.000 3 1.02 6.20 Histidine 59.4617 1.000 4 1.015.20 Histidine 59.4767 1.000 5 1.01 6.20 Histidine 59.4702 1.000 6 1.016.18 Histidine 59.4525 1.000 7 1.01 6.18 Histidine 59.4554 1.000 8 1.005.19 Histidine 59.4736 1.000 9 1.02 5.19 Histidine 59.4524 1.000 10 1.026.19 Histidine 59.4537 1.000 11 1.01 6.20 Histidine 59.4644 1.000 121.02 6.20 Histidine 59.4559 1.000 13 1.01 5.18 Histidine 59.4508 1.00014 1.02 6.19 Histidine 59.4556 1.000 15 1.00 6.21 Histidine 59.485 1.00016 1.01 6.18 Histidine 59.4505 1.000 17 1.02 5.19 Histidine 59.45791.000 18 1.03 6.21 Histidine 59.4523 1.000 19 1.01 6.18 Histidine59.4562 1.000 20 1.01 6.21 Histidine 59.4702 1.000 21 1.00 5.19Histidine 59.4589 1.000 22 1.01 5.20 Histidine 59.4711 1.000 23 1.056.21 Histidine 59.4363 0.997 23 Solutions found Solutions for Succinate1 1.00 5.94 Succinate 57.8819 0.633 2 1.00 5.94 Succinate 57.8819 0.6333 1.00 5.94 Succinate 57.8819 0.633 4 1.00 5.93 Succinate 57.8819 0.6335 1.00 5.95 Succinate 57.8818 0.633 6 1.00 5.92 Succinate 57.8818 0.6337 1.00 5.96 Succinate 57.8817 0.633 7 Solutions found

TABLE 36 Optimization results based on obtained melting temperatures inthe screening of a wide range of excipients in a histidine, acetate orphosphate buffer for the formulation of 23IL0075. The results wereordered from high to low Tm value. The formulation composition has to beread combining the identity of the excipients in columns 2 to 4 and theamount of each excipient in columns 5 to 7. Combination Sugar/ Amino %sugar/ % amino % Tm value number polyol Detergent acid Buffer polyolacid Detergent (° C.) 47 Sucrose Tween 20 Glycine Histidine PH 6 10.5 00 62.97 44 Sucrose Tween 20 Arg/Glu Histidine PH 6 10.5 0 0 62.5 5Mannitol P-F68 Glycine Histidine PH 6 5.6 0 0 62.22 41 Sucrose P-F68Glycine Histidine PH 6 10.5 0 0 62.22 11 Mannitol Tween 20 GlycineHistidine PH 6 5.6 0 0 62.18 46 Sucrose Tween 20 Glycine Acetate PH 5.510.5 0 0 62.16 42 Sucrose P-F68 Glycine Phosphate PH 6 6.3 0.91 0 62.1338 Sucrose P-F68 Arg/Glu Histidine PH 6 10.5 0 0 61.9 6 Mannitol P-F68Glycine Phosphate PH 6 3.7 0.77 0 61.86 43 Sucrose Tween 20 Arg/GluAcetate PH 5.5 10.5 0 0 61.77 40 Sucrose P-F68 Glycine Acetate PH 5.58.8 0.37 0 61.74 22 Sorbitol P-F68 Glycine Acetate PH 5.5 3.9 0.69 061.7 48 Sucrose Tween 20 Glycine Phosphate PH 6 9.2 0.28 0 61.67 4Mannitol P-F68 Glycine Acetate PH 5.5 4.7 0.37 0 61.58 53 Sucrose Tween80 Glycine Histidine PH 6 10.5 0 0 61.58 54 Sucrose Tween 80 GlycinePhosphate PH 6 6.4 0.90 0 61.52 17 Mannitol Tween 80 Glycine HistidinePH 6 5.6 0 0 61.44 37 Sucrose P-F68 Arg/Glu Acetate PH 5.5 10.5 0 061.43 23 Sorbitol P-F68 Glycine Histidine PH 6 5.6 0 0 61.39 12 MannitolTween 20 Glycine Phosphate PH 6 5.6 0 0 61.33 10 Mannitol Tween 20Glycine Acetate PH 5.5 5.6 0 0 61.2 30 Sorbitol Tween 20 GlycinePhosphate PH 6 0.0 2.3 0.0021 61.2 29 Sorbitol Tween 20 GlycineHistidine PH 6 5.6 0 0 61.19 50 Sucrose Tween 80 Arg/Glu Histidine PH 610.5 0 0 61.14 52 Sucrose Tween 80 Glycine Acetate PH 5.5 8.4 0.46 061.1 2 Mannitol P-F68 Arg/Glu Histidine PH 6 5.6 0 0 61.07 45 SucroseTween 20 Arg/Glu Phosphate PH 6 10.5 0 0 61.01 18 Mannitol Tween 80Glycine Phosphate PH 6 5.6 0 0 60.93 8 Mannitol Tween 20 Arg/GluHistidine PH 6 5.6 0 0 60.88 28 Sorbitol Tween 20 Glycine Acetate PH 5.55.6 0 0 60.86 24 Sorbitol P-F68 Glycine Phosphate PH 6 3.2 1.00 0 60.8416 Mannitol Tween 80 Glycine Acetate PH 5.5 5.6 0 0 60.73 19 SorbitolP-F68 Arg/Glu Acetate PH 5.5 5.6 0 0.062 60.71 49 Sucrose Tween 80Arg/Glu Acetate PH 5.5 10.5 0 0 60.68 20 Sorbitol P-F68 Arg/GluHistidine PH 6 5.6 0 0.036 60.65 39 Sucrose P-F68 Arg/Glu Phosphate PH 610.5 0 0 60.5 1 Mannitol P-F68 Arg/Glu Acetate PH 5.5 5.6 0 0 60.43 34Sorbitol Tween 80 Glycine Acetate PH 5.5 3.9 0.69 0 60.29 26 SorbitolTween 20 Arg/Glu Histidine PH 6 5.6 0 0.0004 60.27 35 Sorbitol Tween 80Glycine Histidine PH 6 5.6 0 0 60.18 14 Mannitol Tween 80 Arg/GluHistidine PH 6 5.6 0 0 60.17 3 Mannitol P-F68 Arg/Glu Phosphate PH 6 5.50.06 + 0.05 0 60.15 25 Sorbitol Tween 20 Arg/Glu Acetate PH 5.5 5.6 00.0017 60.04 7 Mannitol Tween 20 Arg/Glu Acetate PH 5.5 5.6 0 0 59.98 51Sucrose Tween 80 Arg/Glu Phosphate PH 6 10.5 0 0 59.98 9 Mannitol Tween20 Arg/Glu Phosphate PH 6 5.6 0 0 59.86 21 Sorbitol P-F68 Arg/GluPhosphate PH 6 5.6 0 0.106 59.73 13 Mannitol Tween 80 Arg/Glu Acetate PH5.5 5.6 0 0 59.54 15 Mannitol Tween 80 Arg/Glu Phosphate PH 6 5.6 0 059.49 36 Sorbitol Tween 80 Glycine Phosphate PH 6 5.6 0 0 59.46 31Sorbitol Tween 80 Arg/Glu Acetate PH 5.5 5.6 0 0.0027 59.36 32 SorbitolTween 80 Arg/Glu Histidine PH 6 4.9 0.34 + 0.29 0.0009 59.34 27 SorbitolTween 20 Arg/Glu Phosphate PH 6 5.6 0 0.0039 59.17 33 Sorbitol Tween 80Arg/Glu Phosphate PH 6 5.6 0 0.0049 58.59

TABLE 37 Comparison of the aggregation onset temperatures and maximumscatter reached for 23IL0075 in 3 buffers (250 μg/mL), as measured byelastic light scattering. (Temperature interval: 45-95° C., temperaturegradient: 2° C./min, data pitch: 1° C., wavelength (ex/em): 500 nm, bandwidth (ex/em): 3 nm.) Maximum Aggregation onset temperature scatterreached 10 mM phosphate 52.1° C. Out of scale pH 6 10 mM acetate pH 651.0° C. Out of scale 10 mM histidine pH 6 52.7° C. 435 abs

TABLE 38 List of buffers tested in freeze/thaw and stir stress study of23IL0075 (10 mg/mL) No. Buffer 1 10 mM Acetate pH 5.5 5.6% mannitol0.0025% Tween 80 2 10 mM Acetate pH 5.5 5.6% mannitol 0.005% Tween 80 310 mM Acetate pH 5.5 5.6% mannitol 0.05% P-F68 4 10 mM Acetate pH 5.55.6% mannitol 0.1% P-F68 5 10 mM Histidine pH 6.0 5.6% mannitol 0.0025%Tween 80 6 10 mM Histidine pH 6.0 5.6% mannitol 0.005% Tween 80 7 10 mMHistidine pH 6.0 5.6% mannitol 0.05% P-F68 8 10 mM Histidine pH 6.0 5.6%mannitol 0.1% P-F68 9 10 mM Phosphate pH 6.0 5.6% mannitol 0.0025% Tween80 10 10 mM Phosphate pH 6.0 5.6% mannitol 0.005% Tween 80 11 10 mMPhosphate pH 6.0 5.6% mannitol 0.05% P-F68 12 10 mM Phosphate pH 6.05.6% mannitol 0.1% P-F68 13 10 mM Acetate pH 5.5 2.8% mannitol 0.0025%Tween 80 1.15% glycine 14 10 mM Acetate pH 5.5 2.8% mannitol 0.005%Tween 80 1.15% glycine 15 10 mM Acetate pH 5.5 2.8% mannitol 0.05% P-F681.15% glycine 16 10 mM Acetate pH 5.5 2.8% mannitol 0.1% P-F68 1.15%glycine 17 10 mM Histidine pH 6.0 2.8% mannitol 0.0025% Tween 80 1.15%glycine 18 10 mM Histidine pH 6.0 2.8% mannitol 0.005% Tween 80 1.15%glycine 19 10 mM Histidine pH 6.0 2.8% mannitol 0.05% P-F68 1.15%glycine 20 10 mM Histidine pH 6.0 2.8% mannitol 0.1% P-F68 1.15% glycine21 10 mM Phosphate pH 6.0 2.8% mannitol 0.0025% Tween 80 1.15% glycine22 10 mM Phosphate pH 6.0 2.8% mannitol 0.005% Tween 80 1.15% glycine 2310 mM Phosphate pH 6.0 2.8% mannitol 0.05% P-F68 1.15% glycine 24 10 mMPhosphate pH 6.0 2.8% mannitol 0.1% P-F68 1.15% glycine

TABLE 39 Stability study of 23IL0075 in 10 mM Histidine pH 6.0 withdifferent excipients. The samples were stressed by 10Xfreeze/thaw, andwere stored at different temperatures (−70° C., 5° C., 25° C. and 37°C.) for a stability study. The stressed and stability samples wereanalyzed using OD measurement, RP-HPLC and SE-HPLC. Conc. Poloxamer No.(mg/mL) Buffer Mannitol Sucrose Glycine 188 Tween-80 1 25 10 mM L- 5.4%0.005% histidine, pH 6 2 25 10 mM L- 10.0% 0.005% histidine, pH 6 3 2510 mM L- 3.5% 3.5% 0.005% histidine, pH 6 4 25 10 mM L- 2.8% 1.15%0.005% histidine, pH 6 5 25 10 mM L- 5.4% 0.05% histidine, pH 6 6 25 10mM L- 10.0% 0.05% histidine, pH 6 7 25 10 mM L- 3.5% 3.5% 0.05%histidine, pH 6 8 25 10 mM L- 2.8% 1.15% 0.05% histidine, pH 6 9 25 10mM L- 3.5% 3.5% histidine, pH 6

TABLE 40 Samples subjected to shear stress by stirring. The samples wereafterwards analyzed by OD measurements, RP-HPLC, SE-HPLC and BIACORE.No. Conc. Buffer Mannitol Sucrose Glycine 1 10 mg/mL 10 mM L-histidine,5.4% pH 6 2 10 mg/mL 10 mM L-histidine, 10.0% pH 6 3 10 mg/mL 10 mML-histidine, 3.5% 3.5% pH 6 4 10 mg/mL 10 mM L-histidine, 2.8% 1.15% pH6

TABLE 41 Results of SE-HPLC and RP-HPLC chromatography of 23IL0075 at 25mg/mL in different candidate formulation buffers (details see tableabove) in accelerated stability study at 25 and 37° C. 3 weeks 37° C. %postpeak % pre-peak in RP-HPLC 6 weeks 37° C. 6 weeks 25° C. in SE-HPLC(N-terminal % pre-peak* % postpeak** % pre-peak* % postpeak** Excipients(oligomers)* pyroglutamate)** in SE-HPLC in RP-HPLC in SE-HPLC inRP-HPLC Mannitol/Tween 80 4.8 6.7 6.9 9.5 0.7 4.9 Sucrose/Tween 80 4.06.0 7.1 9.2 0.6 4.1 Mannitol/sucrose/Tween 80 5.3 5.6 9.5 8.5 0.6 3.8Mannitol/Glycine/Tween 80 7.2 5.6 12   8.8 0.7 3.9 Mannitol/PF 5.0 6.2 12*** 14.7*** 0.8 5.6 Sucrose/PF 3.7 5.8 7.9 9.4 0.6 4.1Mannitol/Sucrose/PF 4.3 5.4 8.5 8.3 0.7 4.1 Mannitol/Glycine/PF 5.4 5.411   8.6 0.8 3.9 Mannitol/Sucrose 3.5 5.2 7.5 8.3 0.8 3.8 *In thereference sample 0.3 to 0.4% pre-peak in SE-HPLC; **% post-peak inRP-HPLC was present; ***The sample stressed for 6 weeks at 37° C. inmannitol and Poloxamer 188 was an outlier also based on the OD500 valuethat was 0.1 compared to OD500 values always below 0.01 for all othersamples.

TABLE 42 Stability data of IL6R304 batch CMC-D-0048, stored at −70° C.Time point (months) Test Method Initial (0) 3 6 Appearance Clear,colorless solution Clear, colorless solution Clear, colorless solutionA280 10.52 mg/mL 10.38 mg/mL 10.45 mg/mL SEC-HPLC Purity = 99.20% Purity= 98.67% Purity = 98.76% Pre peaks = 0.80% Pre peaks = 1.33% Pre peaks =1.24% Post peaks = 0.00% Post peaks = 0.00% Post peaks = 0.00% cIEFPurity = 100.00% Purity = 100.00% Purity = 99.30% Post peak = 0.00% Postpeak = 0.00% Post peak = 0.70% RP-HPLC Purity = 93.90% Purity = 91.93%Purity = 92.8% Pre peak 1 = 0.00% Pre peak 1 = 0.14% Pre peak 1 = 0.12%Pre peak 2 = 3.20% Pre peak 2 = 3.41% Pre peak 2 = 3.00% Post peak 1 =0.00% Post peak 1 = 0.00% Post peak 1 = 0.00% Post peak 2 = 2.60% Postpeak 2 = 4.10% Post peak 2 = 3.60% Post peak 3 = 0.00% Post peak 3 =0.00% Post peak 3 = 0.00% Post peak 4 = 0.20% Post peak 4 = 0.29% Postpeak 4 = 0.32% Post peak 5 = 0.00% Post peak 5 = 0.14% Post peak 5 =0.15% Potency 1.256 ± 0.084 0.973 ± 0.072 1.049 ± 0.090 (IL-6Rihibition) Potency 1.044 ± 0.094 0.955 ± 0.085 0.985 ± 0.069 (HSAbinding)

TABLE 43 Stability data of IL6R304 batch CMC-D-0048, stored at +5° C.Time point (months) Test Method Initial (0) 3 6 Appearance Clear,colorless solution Clear, colorless Clear, colorless solution solutionA280 10.52 mg/mL 10.29 mg/mL 10.33 mg/mL SEC-HPLC Purity = 99.20% Purity= 98.50% Purity = 98.62% Pre peaks = 0.80% Pre peaks = 1.50% Pre peaks =1.38% Post peaks = 0.00% Post peaks = 0.00% Post peaks = 0.00% cIEFPurity = 100.00% Purity = 100.00% Purity = 99.30% Post peak = 0.00% Postpeak = 0.00% Post peak = 0.70% RP-HPLC Purity = 93.90% Purity = 91.71%Purity = 92.30% Pre peak 1 = 0.00% Pre peak 1 = 0.15% Pre peak 1 = 0.11%Pre peak 2 = 3.20% Pre peak 2 = 3.53% Pre peak 2 = 3.20% Post peak 1 =0.00% Post peak 1 = 0.00% Post peak 1 = 0.00% Post peak 2 = 2.60% Postpeak 2 = 4.16% Post peak 2 = 3.90% Post peak 3 = 0.00% Post peak 3 =0.00% Post peak 3 = 0.00% Post peak 4 = 0.20% Post peak 4 = 0.29% Postpeak 4 = 0.35% Post peak 5 = 0.00% Post peak 5 = 0.15% Post peak 5 =0.12% Potency 1.256 ± 0.084 0.959 ± 0.061 1.015 ± 0.076 (IL6Rinhibition) Potency 1.044 ± 0.094 0.930 ± 0.103 0.983 ± 0.078 (HSAbinding)

TABLE 44 Stability data of IL6R304 batch CMC-D-0048, stored at +25° C.Time point (months) Test Method Initial (0) 3 6 Appearance Clear,colorless solution Clear, colorless solution Clear, colorless solutionA280 10.52 mg/mL 10.29 mg/mL 10.48 mg/mL SEC-HPLC Purity = 99.20% Purity= 97.81% Purity = 97.13% Pre peaks = 0.80% Pre peaks = 1.57% Pre peaks =1.87% Post peaks = 0.00% Post peaks = 0.62% Post peaks = 1.00% cIEFPurity = 100.00% Purity = 96.40% Purity = 92.30% Post peak = 0.00% Postpeak = 3.60% Post peak = 7.70% RP-HPLC Purity = 93.90% Purity = 87.77%Purity = 82.30% Pre peak 1 = 0.00% Pre peak 1 = 0.44% Pre peak 1 = 0.87%Pre peak 2 = 3.20% Pre peak 2 = 4.56% Pre peak 2 = 6.40% Post peak 1 =0.00% Post peak 1 = 0.00% Post peak 1 = 1.10% Post peak 2 = 2.60% Postpeak 2 = 6.26% Post peak 2 = 8.80% Post peak 3 = 0.00% Post peak 3 =0.54% Post peak 3 = 0.86% Post peak 4 = 0.20% Post peak 4 = 0.30% Postpeak 4 = 0.32% Post peak 5 = 0.00% Post peak 5 = 0.13% Post peak 5 =0.16% Potency 1.256 ± 0.084 0.945 ± 0.065 0.949 ± 0.066 (IL6Rinhibition) Potency 1.044 ± 0.094 0.967 ± 0.095 0.926 ± 0.065 (HSAbinding)

Example 4 Generation of NFDs 4.1 Fermentation of Polypeptide A (SEQ IDNO: 71 Producing E. coli Clone

Fermentation of Polypeptide A (SEQ ID NO: 7) clone 1 (identified asdisclosed in WO 2006/122825) was carried out at 10 liter scale inTerrific Broth (Biostat Bplus, Sartorius) with 100 μg/ml carbenicillin.A two percent inoculum of the preculture (grown overnight in TB, 2%glucose, 100 μg/ml carbenicillin) was used to start the productionculture (22° C./1 vvm). Induction (using 1 mm IPTG) was started at anOD₆₀₀ of 8.0. After a short induction at 22° C., the cell paste wascollected via centrifugation (Sigma 8K, rotor 12510; 7000 rpm for 30min) and frozen at −20° C.

4.2 Purification of Polypeptide A

Purified Polypeptide A (monomer and dimer) was generated via a processconsisting of 6 steps:

4.2.1 Extraction from Cell Pellet

The frozen cell pellet was thawed, the cells were resuspended in coldPBS using an Ultra Turrax (Ika Works; S25N-25G probe, 11.000 rpm.) andagitated for 1 h at 4° C. This first periplasmic extract was collectedvia centrifugation; a second extraction was carried out in a similar wayon the obtained cell pellet. Both extractions did account for more than90% of the periplasmic Polypeptide A content (the 2^(nd) extraction didyield about 25%).

4.2.2 Removal of Major Contaminants Via Acidification

The periplasmic extract was acidified to pH=3.5 using 1M citric acid(VWR (Merck) #1.00244.0500) 10 mM molar final pH=3.5 and further pHadjusted with 1M HCl. The solution was agitated overnight at 4° C. Theprecipitated proteins and debris was pelleted down via centrifugation.

4.2.3 Micro-Filtration and Concentration of the Extract

The supernatant was made particle free using a SARTOCON Slice Crossflowsystem (17521-101, Sartorius) equipped with Hydrosart 0.20 μm membrane(305186070 10-SG, Sartorius) and further prepared for Cation ExchangeChromatography (CEX) via Ultra filtration. The volume that needed to beapplied to CEX was brought down to approx 2 liter via ultra filtrationusing a SARTOCON Slice Crossflow system equipped with Hydrosart10,000MWCO membranes (305144390 1E-SG, Sartorius). At that point theconductivity (<5 mS/cm) and pH (=3.5) were checked.

4.2.4. Capture and Purification Via CEX

The cleared and acidified supernatant was applied to a Source 305 column(17-1273-01, GE Healthcare) equilibrated in buffer A (10 mM Citric acidpH=3.5) and the bound proteins were eluted with a 10CV linear gradientto 100% B (1M NaCl in PBS). The Polypeptide A fraction was collected andstored at 4° C.

4.2.5. Affinity Purification on Protein a Column

Polypeptide A (amount=well below column capacity) was further purifiedvia Protein A affinity chromatography (MabSelect Xtra®, 17-5269-07, GEHealthcare). A one step elution was carried out using 100 mM Glycine pH2.5. The collected sample was immediately neutralized using 1M TrispH7.5 (see FIG. 58).

4.2.6. Size Exclusion Chromatography (Optional e.g. in Order to IsolateNFDs and/or Determine Amount of NFDs)

The purified Nanobody® fraction was further separated and transferred toD-PBS (Gibco#14190-169) via SEC using a Hiload™ XK26/60 SUPERDEX 75column (17-1070-01, GE Healthcare) equilibrated in D-PBS. Fraction 2contained the dimeric Polypeptide A (see FIG. 59).

In a further experiment, Polypeptide A (SEQ ID NO: 7) was accumulated ona Protein A column, its concentration well above 5 mg polypeptide A/mlresin, and eluted via a steep pH shift (one step buffer change to 100 mMGlycine pH 2.5). During elution of the polypeptide A from the column itwas ‘stacked’ into an elution front, consisting of ‘locally’ very highconcentrations (actual value after elution>5 mg/ml), and combinationwith the pH shift led to the isolation of about 50% stable dimer (seeFIG. 54).

The shift from monomer to dimer is demonstrated via size exclusionchromatography (SEC), allowing determination of the percentage ofdimerization (see FIG. 55). When loading less polypeptide A on Protein A(i.e. 2 mg/ml resin under otherwise same conditions as above, i.e. onestep elution with 100 mM Glycine pH 2.5), almost no dimers (<5%) weredetected during SEC (see FIG. 56 and FIG. 57). Similarly, NFDs of apolypeptide comprising one singe variable domain (NFD-Mo), a polypeptidecomprising three single variable domains (NFD-Tri), and a polypeptidecomprising a HSA (human serum albumin) and a single variable domainfusion were obtained (see Table 45).

TABLE 45 Examples of obtained NFDs Code for SEQ ID NO of IsolatedMonomeric monomeric stable NFD Monomeric polypeptide polypeptidebuilding block Obtained by type comprising Polypeptide A 7 Protein A +SEC NFD-Di Two identical single variable domains Polypeptide B, 8 IMAC +AEX + SEC; NFD-Mo One single variable domain also referred Proteinbinding to human serum to as Alb11 A + SEC albumin Polypeptide C 9Protein A + SEC NFD-Tri Three single variable domains of which one bindsto human serum albumin and the two other single variable domains bind toa receptor target Polypeptide D 10 Protein A + SEC NFD-Mo Singe variabledomain and HSA Polypeptide E 11 Protein A + SEC NFD-Di Two singlevariable domains of which one binds to human serum albumin and the othersingle variable domain binds to a receptor target Polypeptide F 12Protein A + SEC NFD-Mo One single variable domain binding to human serumalbumin

Example 5 Stability of NFDs

During purification of Polypeptide A stable non fused dimers (NFDs) weregenerated (see above). In order to get more insight into the stabilityand nature of this non-covalent interaction, stable Polypeptide A NFDswere subjected to distinctive conditions aiming to dissociate the dimerinto monomer. The stability of the complex was evaluated via 3 criteria:heat-stability, pH-stability, organic solvent resistance andcombinations thereof.

51 Experimental Set Up

The Polypeptide A NFD was generated during a Polypeptide A preparation(see above) and was stored at −20° C. for 2.5 years. This dimericmaterial was obtained via Protein A chromatography and Size ExclusionChromatography (SEC) in PBS. In the latter, monomeric and dimericmaterial were separated to a preparation of >95% pure dimer. Uponthawing about 5% monomeric material was detected (see arrow in FIG. 60).The concentration of dimeric material was 0.68 mg/ml.

Analytic Size Exclusion Chromatography

The stability of the Polypeptide A NFD dimer was analysed via analyticSEC on a SUPERDEX 75 10/300GL column (17-5174-01, GE Healthcare) usingan Äkta Purifier10 workstation (GE Healthcare). The column wasequilibrated in D-PBS at room temperature (20° C.). A flow rate of 1ml/min was used. Proteins were detected via absorption at 214 nm. 12 μgsamples of Polypeptide A NFD were injected.

Overview Analytic SEC Runs:

-   -   20 μl POLYPEPTIDE A NFD+90 μl D-PBS→15′/50° C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl D-PBS→15′/20° C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl D-PBS→30′/45° C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl D-PBS→15′/60° C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl D-PBS→15′/70° C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [100 mM Piperazin pH=10.2]→ON/4°        C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [100 mM Glycin pH=2.5]→ON/4°        C.→100 μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [10% Isopropanol]→ON/4° C.→100 μl        analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [30% Isopropanol]→ON/4° C.-100 μl        analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [1% TFA]→15′/20° C.→100 μl        analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [30% Isopropanol]→15′/50° C.-100        μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [30% Isopropanol]→15′/20° C.-100        μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [30% Isopropanol]→15′/40° C.-100        μl analyzed    -   20 μl POLYPEPTIDE A NFD+90 μl [30% Isopropanol]→15′/45° C.-100        μl analyzed

This material was used in several experiments: 20 μl dimer fractionswere diluted with 90 μl D-PBS or other solvents, incubated underdifferent conditions and 100 μl samples were analysed via analytic SEC.

5.2 Tests

In a first set of experiments incubation during 15 minutes at increasingtemperatures was carried out (45, 50, 60 and 70° C.), followed byanalytic SEC (SUPERDEX 75™ 10/300GL). An incubation at 70° C. during 15min resulted in an almost complete shift to monomeric Polypeptide A,whereas lower temperatures (e.g. 50° C.) did not result in such adrastic effect. After 15 minutes at 60° C. about 25% dissociatedmaterial was detected (see FIG. 60).

In a second set of experiments the effect of pH on the stability ofPolypeptide A NFD was explored. 20 μl NFD was mixed with 90 μl [100 mMPiperazin pH=10.2] or 90 μl [100 mM Glycine, pH=2.5] and incubatedovernight (ON) at 4° C. 20 μl NFD was mixed with 90 μl [1% TFA] at roomtemperature for 15 minutes and then immediately analysed via SEC. Thecontrol was incubated in D-PBS. Samples were analysed via SEC the nextday (see FIG. 61).

A third set of experiments consisted of a combined treatment:Temperature and organic solvent (Isopropanol). Neither incubation in 10or 30% Isopropanol overnight at 4° C., nor incubation in 10 or 30%Isopropanol during 15 minutes at room temperature resulted in anysignificant dissociation. However, combining increased temperatures andorganic solvent resulted in a much faster dissociation into monomer.Whereas incubation at 45° C. or 30% Isopropanol had no effect alone,combining both (during 15 minutes) resulted in an almost fulldissociation into monomer. Isopropanol treatment at 40° C. yielded only30% dissociation (see FIG. 62).

5.3 Discussion

The concentration independent character of the dimer/monomer equilibriumwas further substantiated by the near irreversibility of the interactionunder physiological conditions. In addition, the rather drastic measuresthat needed to be applied to (partly) dissociate the dimer into monomerpoint to an intrinsic strong interaction. Dissociation is only obtainedby changing the conditions drastically (e.g. applying a pH below 2.0) orsubjecting the molecule to high energy conditions. Temperature stabilitystudies (data not shown) indicate that the Tm of Polypeptide A NFD is73° C., so the observed dissociation into monomer might be indeed linkedto (partial) unfolding.

The solubilizing properties of TFA combined with protonation at extremelow pH, increasing the hydrophilicity, also results in dissociation.

The combination of elevated temperature and organic solvent dissociationindicates that the interaction is mainly based on e.g. hydrophobicity(e.g. Van der Waals force), hydrogen bonds, and/or ionic interactions.

The conditions used to drive these dimers apart may be also useful toexplore when determining further methods for producing these dimers,i.e. combining these procedures (e.g. temperature of higher than 75degrees Celsius) with a high polypeptide concentration.

Example 6 Ligand Binding of NFDs

The binding of Ligand A (SEQ ID NO: 13) to Polypeptide A and PolypeptideA NFD-Di was studied via analytic size exclusion.

6.1 Ligand A Production

Ligand A is known to be the binding domain of Polypeptide A, i.e. itcomprises the epitope of Polypeptide A (i.e. Ligand A represents the A1domain of vWF).

Ligand A [1.46 mg/ml] was produced via Pichia in shaker flasks. Biomasswas produced in BGCM medium. For induction a standard medium switch tomethanol containing medium (BMCM) was done. The secreted protein wascaptured from the medium via IMAC, further purified on a Heparinaffinity column and finally formulated in 350 mM NaCl in 50 mM Hepes viaSize Exclusion Chromatography (SEC) (SUPERDEX 75 HiLoad 26/60).

6.2 Analytic SEC on SUPERDEX 200 10/300GL

Polypeptide A (with 2 expected binding sites) and its corresponding NFD(with 4 expected binding sites) were obtained as disclosed in example 4and added to 5× excess of the Ligand A. The resulting shift in molecularweight was studied via size exclusion chromatography (SEC) (FIG. 63).The shift in retention approximately indicates the number of Ligand Amolecules binding to the Polypeptide A or corresponding NFD. Ligand Ahas a molecular weight of about 20 kDa. The molecular weight shift ofthe NFD/Ligand A complex compared to NFD alone or Polypeptide/Ligand Acomplex to Polypeptide A indicates the number of Ligand A per NFD or perPolypeptide A bound (see Table 46).

TABLE 46 Molecular weight shift of the NFD/Ligand A complex compared toNFD alone or Polypeptide/Ligand A complex to Polypeptide A Measured MWshift Estimated Measured with Number of Retention MW Theoretical MWligand A Ligand A Material (ml) (KDa)* (Da) exposure bound NFD + LigandA 13.2 123.6 153940 (assuming 4 62.5 3 Ligand A bindings) PolypeptideA + 14.1 79.1 76970 (assuming 2 54.1 2 ligand A Ligand A bindings) NFD14.7 61.1 (55752) Not Not applicable applicable Polypeptide A 16.6 25.0(27876) Not Not applicable applicable Ligand A 16.8 22.8 (24547) Not Notapplicable applicable *MW was calculated based on curve fitting ofMolecular weight standards (Biorad #151-1901) run on the same columnunder same conditions (see FIG. 64).

6.3 Overview Analytic SEC Runs on SUPERDEX 75 10/300GL

(B7) 040308.1: Complex ligand-NFD 5 μl mix (ON stored at 4° C.)+80 μl Abuffer(B7) 040308.2: 20 μl Molecular weight marker+80 μl A buffer(B7) 040308.3: Complex 20 μl ligand+90 μl A buffer, 4 h atRT+Polypeptide A [17 μl 1/10], 30 min at RT before analysis(B7) 040308.4: Polypeptide A [17 μl in 90 μl A buffer](B7) 040308.5: Ligand in A buffer (1 h at RT)+Polypeptide A, 15 min atRT before analysis.

(B7) 040308.6: Ligand+Buffer A+NFD

(B7) 040308.7: rest sample #6 after 1 h at RT

(B7) 040308.8: Buffer A+NFD

The correlation of the expected MW shows that more than 2 ligands(likely 3 and possibly 4 due to the atypical behaviour of Ligand Acomplexes on the SEC) are bound by the NFD.

Example 7 Further Characterization of a NFD with Polypeptide B Example7.1 Crystal Structure of a Non-Fused Dimer: Polypeptide B 7.1.1Crystallization

The protein was first concentrated to a concentration of about 30 mg/mL.The purified protein was used in crystallization trials withapproximately 1200 different conditions. Conditions initially obtainedhave been optimized using standard strategies, systematically varyingparameters critically influencing crystallization, such as temperature,protein concentration, drop ratio and others. These conditions were alsorefined by systematically varying pH or precipitant concentrations.

7.1.2 Data Collection and Processing

Crystals have been flash-frozen and measured at a temperature of 100K.The X-ray diffraction data have been collected from the crystals at theSWISS LIGHT SOURCE (SLS, Villingen, Switzerland) using cryogenicconditions.

The crystals belong to the space group P 2₁ with 2 molecules in theasymmetric unit. Data were processed using the program XDS and XSCALE.Data collection statistics are summarized in Table 47.

TABLE 47 Statistics of data collection and processing X-ray source PX-3(SLS¹) Wavelength (Å)   0.97800 Detector MARCCD Temperature (K)  100Space group P 2₁ Cell dimensions: a; b; c (Å) 37.00; 67.06; 41.14 α; β;γ (°) 90.0; 97.7; 90.0 Resolution (Å)²   1.20 (1.30-1.26) Uniquereflections² 60716 (4632) Multiplicity²   4.1 (4.1) Completeness (%)²  97.7 (96.7) R_(sym) (%)^(2,3)   7.2 (41.4) R_(meas) (%)^(2,4)   8.3(47.6) I/σ² —(—) Mean(I)/sigma^(2,5)   12.83 (4.01) ¹SWISS LIGHT SOURCE(SLS, Villingen, Switzerland) ²Numbers in brackets corresponds to theresolution bin with R_(sym) = 41.4% $\begin{matrix}{{{}_{}^{}{}_{}^{}} = \frac{\sum\limits_{h}{\sum\limits_{i}^{n_{h}}{{{\hat{I}}_{h} - I_{h,i}}}}}{\sum\limits_{h}{\sum\limits_{i}^{n_{h}}I_{h,i}}}} \\{{{{with}\mspace{14mu} {\hat{I}}_{h}} = {\frac{1}{n}{\sum\limits_{i}^{n_{h}}I_{h,i}}}},{{where}\mspace{14mu} I_{h,i}\mspace{14mu} {is}\mspace{14mu} {the}\mspace{14mu} {intensity}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {ith}}} \\{{measurement}\mspace{14mu} {of}\mspace{14mu} h}\end{matrix}\quad$ $\begin{matrix}{{{}_{}^{}{}_{}^{}} = \frac{\sum\limits_{h}{\sqrt{\frac{n_{h}}{n_{h} - 1}}{\sum\limits_{i}^{n_{h}}{{{\hat{I}}_{h} - I_{h,i}}}}}}{\sum\limits_{h}{\sum\limits_{i}^{n_{h}}I_{h,i}}}} \\{{{{with}\mspace{14mu} {\hat{I}}_{h}} = {\frac{1}{n}{\sum\limits_{i}^{n_{h}}I_{h,i}}}},{{where}\mspace{14mu} I_{h,i}\mspace{14mu} {is}\mspace{14mu} {the}\mspace{14mu} {intensity}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {ith}}} \\{{measurement}\mspace{14mu} {of}\mspace{14mu} h}\end{matrix}\quad$ ⁵Calculated from independent reflections

7.1.3 Structure Modelling and Refinement

The phase information necessary to determine and analyze the structurewas obtained by molecular replacement.

Subsequent model building and refinement was performed according tostandard protocols with the software packages CCP4 and COOT. For thecalculation of the R-factor, a measure to cross-validate the correctnessof the final model, 1.6% of measured reflections were excluded from therefinement procedure (Table 48). The ligand parameterisation was carriedout with the program CHEMSKETCH. LIBCHECK (CCP4) was used for generationof the corresponding library files. Statistics of the final structureand the refinement process are listed in Table 48.

TABLE 48 Refinement statistics¹ Resolution (Å) 20.0-1.20 Number ofreflections 59743/972 (working/test) R_(cryst) (%) 14.8 R_(free) (%)16.9 Total number of atoms in protein 1759 Deviation from idealgeometry² Bond lengths (Å) 0.006 Bond angles (°) 1.17 ¹Values as definedin REFMAC5, without sigma cut-off ²Root mean square deviations fromgeometric target values

7.1.4 Overall Structure

The asymmetric unit of crystals is comprised of 2 monomers. TheNanobody® is well resolved by electron density maps.

7.1.5 Structure

The 2 polypeptide B-monomers that form the polypeptide B dimer (NFD-Mo)have a properly folded CDR1 and CDR2 and framework 1-3. The framework 4residues (residues 103-113 according to the Kabat numbering scheme) areexchanged between the 2 monomers. This results in an unfolded CDR3 ofboth monomers that are present in the dimer (see FIG. 65). Dimerformation is mediated by the exchange of a β-strand from Q105 to Ser113between both monomers (see FIG. 66). Strand exchange is completelydefined by electron density (see FIG. 67).

The residues of framework 1-3 and CDR1 and CDR2 of the monomer that formthe dimer have a classical VHH fold and are almost perfectlysuperimposable on a correctly folded polypeptide B VHH domain (backbonermsd<0.6 Å). A decreased stabilization of CDR3 in polypeptide B comparedto the structures of VHH's with similar sequences to polypeptide B canbe one of the causes of the framework 4 exchanged dimerization. Aslightly modified form of polypeptide B with a Proline at position 45shows a hydrogen-bond between Y91 and the main-chain of L98. Thishydrogen-bond has a stabilizing effect on the CDR3 conformation.

Due to the leucine at position 45 in polypeptide B, the tyrosine 91 cannot longer form the hydrogen-bond with the main-chain of leucine-98.This leads to a decreased stabilization of the CDR3 conformation inpolypeptide B (FIG. 68).

Example 7.2 Stability and Various Other Studies of the NFD withPolypeptide B 7.2.1 Production and Isolation of Polypeptide B

Tagless polypeptide B was over-expressed in E. coli TOP10 strain at 28°C. after overnight induction with 1 mM IPTG. After harvesting, thecultures were centrifuged for 30 minutes at 4500 rpm and cell pelletswere frozen at −20° C. Afterward the pellets were thawed andre-suspended in 50 mM phosphate buffer containing 300 mM NaCl and shakenfor 2 hours at room temperature. The suspension was centrifuged at 4500rpm for 60 minutes to clear the cell debris from the extract. Thesupernatant containing polypeptide B, was subsequently loaded on PorosMABCAPTURE A column mounted on Akta chromatographic system. Afterwashing the affinity column extensively with D-PBS, bound polypeptide Bprotein was eluted with 100 mM Glycine pH 2.7 buffer. Fractions elutedfrom column with acid were immediately neutralized by adding 1.5M TRISpH8.5 buffer. At this stage the protein was already very pure as only asingle band of the expected molecular weight was observed onCoomassie-stained SDS-PAGE gels. The fractions containing thepolypeptide B were pooled and subsequently concentrated byultrafiltration on a stirred cell with a polyethersulphone membrane witha cut-off of 5 kDa (MILLIPORE). The concentrated protein solution wasafterwards loaded on a SUPERDEX 75 XK 26/60 column. On the chromatogram(see FIG. 69), besides the main peak eluting between 210 mL and 240 mL,a minor peak eluting between 180 mL and 195 ml was present.

Analysis on SDS-PAGE uncovered that both major peaks contain a singlepolypeptide with the same mobility (data not shown). This observationwas the first indication that the peak eluting between 180 mL and 195 mLis a dimeric species, whereas the material eluting between 210 mL and240 mL is a monomer. Further analysis on reversed phase chromatographyand LC/MS of the dimeric and monomer species uncovered that both containthe same polypeptide with a molecular weight of about 12110 dalton. Inthis way from a 10 L fermentor run, in total 30 mg of the dimericspecies and 1200 mg of the monomeric form of polypeptide B was isolated.

7.2.2 Antigen Binding Properties

The binding of the polypeptide B monomer and Polypeptide B dimer tohuman serum albumin was tested by surface plasmon resonance in a BIACORE3000 instrument. In these experiments human serum albumin wasimmobilized on CM5 chip via standard amine coupling method. The bindingof both monomeric polypeptide B and dimeric polypeptide B at aconcentration of 10 nanomolar were tested. Only for the monomer, bindingwas observed whereas no increase in signal was observed for the dimericpolypeptide B.

7.2.3 Difference in Physicochemical Properties Between Monomeric andDimeric Polypeptide B

The fluorescent dye SYPRO orange (5000× Molecular Probes) can be used tomonitor the thermal unfolding of proteins or to detect the presence ofhydrophobic patches on proteins. In the experiment, monomeric anddimeric Polypeptide B at a concentration of 150 microgram/mL were mixedwith SYPRO orange (final concentration 10×). The solution was afterwardstransferred to quartz cuvette, and fluorescence spectra were recorded onA Jasco FP6500 instrument. Excitation was at 465 nm whereas the emissionwas monitored from 475 to 700 nm. As shown in FIG. 70, only a strongsignal for the dimeric polypeptide B was observed, whereas no increasein fluorescence emission intensity was observed for the polypeptide Bmonomeric species. This observation strongly suggests that monomeric anddimeric forms of polypeptide B have a distinct conformation.

7.2.4 AUC-EQ—Sedimentation-Diffusion Equilibrium Material and Methods

Experiments were performed with an Analytical ultracentrifuge XL-I fromBeckman-Coulter using the interference optics of the instrument. Datawere collected at a temperature of 20° C. and rotational speeds of 25000rpm and 40000 rpm. 150 μL were filled in the sample sector of 12 mm twosector titanium centerpieces. Samples were diluted with standard PBS,which was also used for optical referencing. Attainment of apparentchemical and sedimentation equilibrium was verified by comparingconsecutive scans until no change in concentration with time wasobserved. Data were evaluated with the model-independent M*-function andvarious explicit models using NONLIN. Standard values for the v of theprotein and the density of the solvent were used. Where appropriate, 95%confidence limits are given in brackets.

Result

Polypeptide B was found to have a molar mass of 11.92 kg/mole(11.86-11.97) kg/mole from a fit assuming a single, monodisperecomponent. This agrees well with the result from the model-free analysiswhich is 12.25 kg/mole at zero concentration. Attempts to describe thedata assuming self-association, non-ideality or polydispersity did notimprove the global rmsd of the fit.

Polypeptide B was equally well-defined, having a molar mass of 23.06kg/mole (22.56-23.44) kg/mole based on a direct fit assuming a single,monodispere component. The model-free analysis revealed a molar mass of22.69 kg/mole. A small contribution from thermodynamic non-idealityimproved the fit slightly but did not alter the molar mass. No evidencefor a reversible self-association could be found.

The ratio of the M(Polypeptide B-dimer)/M(Polypeptide B) was 1.93. Thesmall deviation from the expected factor of 2 can be explained by adifferent v of Polypeptide B Dimer compared to Polypeptide B, slightdensity differences for the different dilutions due to the slightlydifferent Polypeptide B, slight density differences for the dilutionsdue to the slightly different buffers used (PBS for dilution and D-PBSfor the stock solutions) and a contribution from non ideality too smallto be reliably described with the data available.

7.2.5 Stability Study of Polypeptide F and Polypeptide B at 4° C., 25°C. and 37° C.

Solutions of monomeric polypeptide F and polypeptide B, formulated inD-PBS, were concentrated to 20 mg/mL and put on storage at 4° C., 25° C.and 37° C. After 3 and 6 weeks samples were analyzed by size exclusionchromatography on a Phenomenex BIOSEP SEC 5-2000 column. In the SECchromatograms of both polypeptide F and Polypeptide B, the presence of apre-peak was only observed in the chromatograms of the samples stored at37° C. The pre-peak corresponding to a dimer, was not observed insamples stored at 4° C., 25° C. or in a reference material stored at−20° C.

In the Table 49 below the percentage of dimer present in the samplesstored at 37° C. (expressed as percentage of area of dimer versus totalarea) for both polypeptide F and polypeptide B are compiled. As can beobserved in this table, it appears that polypeptide B is moresusceptible to dimer formation than polypeptide F.

TABLE 49 Nanobody ® % dimer-3 weeks % dimer-6 weeks Polypeptide F 3.15.8 Polypeptide B 20.9 37.1

In a separate experiment the effect of mannitol as excipient in theformulation buffer was evaluated. In this case monomeric polypeptide Bwas formulated at a protein concentration of 18 mg/mL respectively inD-PBS or D-PBS containing 5% mannitol. Samples were stored at 37° C. andanalyzed by size exclusion chromatography on a Phenomenex BIOSEP SECS-2000 column after 2, 4, 6 and 8 weeks.

In the table 50 below, the percentage of dimer present in the samplesstored at 37° C. (expressed as percentage of area of dimer versus totalarea) for Polypeptide B stored in D-PBS and in D-PBS/5% mannitol werecompiled. As shown in this table, the presence of mannitol in the bufferhad a clear effect on the kinetics of dimer formation of polypeptide Bat 37° C.

TABLE 50 % dimer % dimer % dimer after 2 after 4 after 6 % dimer after 8weeks weeks weeks weeks Polypeptide B 13.5 22.1 30.0 41.8 Polypeptide Bwith 5.3 11.7 16.8 23.7 5% mannitol

In another experiment, solutions of both monomeric polypeptide F andpolypeptide B at concentrations of 5 mg/ml, 10 mg/mL and 20 mg/mL inD-PBS were stored at 37° C. After 6 weeks, samples were analyzed by sizeexclusion chromatography on a Phenomenex BIOSEP SEC S-2000 column. Inthe table below the percentage of dimer present in the samples stored at37° C. (expressed as percentage of area of dimer versus total area) forpolypeptide F and polypeptide B stored at 5 mg/mL, 10 mg/mL and 20 mg/mLare compiled. From this experiment we learned, as observed earlier, thatdimer formation proceeds faster for the polypeptide B than forpolypeptide F, but also that the kinetics of dimer formation are largelydependent on the protein concentration.

TABLE 51 % % dimer % dimer (5 mg/mL) dimer (10 mg/mL) (20 mg/mL)Polypeptide F 1.2 3.1 5.7 Polypeptide B 13.0 20.6 36.9

Similarly, dimer and possibly multimer formation was observed forpolypeptides comprising polypeptide B and other single variable domains,e.g. polypeptides comprising one polypeptide B and 2 Nanobodies® bindingto a therapeutic target (e.g. 2 identical Nanobody® directed against atherapeutic target). The dimer/multimer formation of said polypeptidescomprising e.g. polypeptide B and other Nanobodies® could be slowed downor in some instances almost avoided if they were formulated in amannitol containing liquid formulation.

Other polyols and/or sugars that are believed to be beneficial to reduceor avoid the formation of dimers (NFDs) and other possibly highermultimers are listed in Table 52. A wide variety of liquid formulationsmay be useful which may consist of or comprise any buffering agent, abiologically effective amount of polypeptide of the invention, aconcentration of mannitol that is no greater than approximately 0.6M andother excipients including polyols, non-reducing sugars, NaCl or aminoacids.

TABLE 52 Polyols sorbitol, mannitol, xylitol, ribitol, erythritolNon-reducing sugars sucrose, trehalose

7.2.6 Chaotrope Induced Unfolding of Polypeptide B and Polypeptide BDimer

Chaotrope induced unfolding is a technique frequently used to assess thestability of proteins. To monitor chaotrope induced unfolding intrinsicfluorescence of tryptophan or tyrosine residue can be used. As unfoldingparameter the ‘center of spectral mass’ (CSM=Σ(fluorescenceintensity×wavenumber)/Σ(fluorescence intensity) can be used. Unfoldingexperiments with Polypeptide B monomer and Polypeptide B dimer wereperformed at 25 μg/mL in Guanidinium Hydrochloride solution in theconcentration range 0-6M. After overnight incubation of these solutionsfluorescence spectra were recorded using a Jasco FP-6500 instrument.Excitation was at 295 nm and spectra were recorded between 310 to 440nm. Using the spectral data the CSM-value was calculated using theformula above. In the FIG. 71, the CSM as a function of GuanidiniumHydrochloride concentration is shown. As can be observed in FIG. 71,polypeptide B dimer unfolds at higher concentrations of GuanidiniumHydrochloride, and allows us to conclude that the monomer is less stablethan the Polypeptide B-dimer.

Example 8 Further Characterization of a NFD with Polypeptide G and H

Different mutants of polypeptide F have been constructed, expressed andpurified. Sequence information is provided below. Purity was analysed ona Coomassie stained gel (FIG. 72) and western blot.

8.1 Binding to Serum Albumin in BIACORE

Binding of Nanobodies® to human serum albumin (HSA) is characterized bysurface plasmon resonance in a BIACORE 3000 instrument, and anequilibrium constant K_(D) was determined. In brief, HSA was covalentlybound to CM5 sensor chips surface via amine coupling until an increaseof 500 response units was reached. Remaining reactive groups wereinactivated. Nanobody® binding was assessed using series of differentconcentrations. Each Nanobody® concentration was injected for 4 min at aflow rate of 45 μl/min to allow for binding to chip-bound antigen. Next,binding buffer without Nanobody® was sent over the chip at the same flowrate to allow dissociation of bound Nanobody®. After 15 minutes,remaining bound analyte was removed by injection of the regenerationsolution (50 mM NaOH).

From the sensorgrams obtained (FIG. 73) for the different concentrationsof each analyte. K_(D) values were calculated via kinetic data analysis.Polypeptide H (with introduction of GL instead of EP, in particular P isreplaced by L, see also FIG. 68 and examples above) had a greater koffrate.

TABLE 53 k_(off) values of Polypeptide F and the humanized derivativesPolypeptide G and Polypeptide H as determined in BIACORE for binding toHSA. Nanobody ® K_(off) (1/s) Polypeptide F 6.83E−4 Polypeptide G1.18E−3 Polypeptide H 1.97E−3

8.2 Stability on Storage

Solutions of monomeric Polypeptide G and Polypeptide H, formulated inD-PBS, are concentrated to 20 mg/mL and put on storage at 4° C., 25° C.and 37° C. After 3 and 6 weeks samples are analyzed by size exclusionchromatography on a Phenomenex BIOSEP SEC S-2000 column.

Example 9 Stability of the Polypeptide I in Different Buffers whenStored at 37° C. Up to 10 Weeks

Polypeptide I (SEQ ID NO: 17) is a trivalent bispecific Nanobodyconsisting of three humanized variable domains of a heavy-chain llamaantibody, of which two identical subunits are specific for binding toRANKL while the remaining subunit binds to HSA.

Polypeptide I was expressed in Pichia pastoris and purified on SPSEPHAROSE as a capturing step and a Q filter as a polishing step or onSP SEPHAROSE as a capturing step and CAPTO MMC as a polishing step oralternatively by using a ProtA capture step followed by and SP SEPHAROSEpolishing step. Concentration of the Polypeptide I and buffer switch toPBS, 10 mM phosphate+100 mM NaCl, 10 mM phosphate+10% mannitol or 10 mMphosphate+50 mM NaCl or others buffers was done via UF/DF or bydialysis. A final filtration on a 0.22 μm filter was performed.Polypeptide I was formulated in different buffers at ˜60 mg/mL (buffers1-12 given in Table 53-A)

TABLE 53-A Overview of the different formulation buffers of PolypeptideI used in stability testing. Concen- tration Polypep- Man- tide I [NaCl]nitol Buffer (mg/mL) Buffer (mM) % (w:v) 1 60 10 mM NaH₂PO₄•2H₂O, pH 750 0 2 60 10 mM NaH₂PO₄•2H₂O, pH 7 100 0 3 60 10 mM NaH₂PO₄•2H₂O, pH 7 010 4 59 10 mM Na-acetate, pH 5.5 50 0 5 59 10 mM Na-acetate, pH 5.5 1000 6 59 10 mM Na-acetate, pH 5.5 0 10 7 60 20 mM L-histidine, pH 5.5 50 08 60 20 mM L-histidine, pH 5.5 100 0 9 60 20 mM L-histidine, pH 5.5 0 1010 58 20 mM L-histidine, pH 6 50 0 11 58 20 mM L-histidine, pH 6 100 012 58 20 mM L-histidine, pH 6 0 10

The stability of the different samples was assessed in acceleratedstress conditions at 37° C.±3° C. Samples were taken after 2, 3, 5 and10 weeks storage at this temperature and were analyzed using SE-HPLC.BIACORE was performed on the samples stored for 10 weeks to evaluateloss in potency.

9.1 SE-HPLC Analysis

The SE-HPLC assay consisted of a pre-packed silica gel TSK-GELG2000SW_(XL) column, a mobile phase consisting of KCl, NaCl andphosphate buffer pH 7.2 (D-PBS) and UV detection at 280 nm. The relativeamount of specific protein impurity was expressed as area %, and wascalculated by dividing the peak area corresponding to the specificprotein or protein impurity by the total integrated area.

The results of the analysis of a sample by SE-HPLC is given in FIG. 74where an example is shown for the sample stored during two weeks at 37°C. in the presence of 50 or 100 mM salt or 10% mannitol-containingphosphate buffer. Storage at 37° C. resulted in the formation of a clearprepeak eluting at about 40 minutes and some minor postpeaks close tothe main peak; these postpeaks elute between 48-55 minutes (see insertin FIG. 74) and represent some degradation fragments. In Table 54 theintegration data for all samples analysed is summarized for thedifferent peaks observed (except buffer peaks after 60 minutes elutiontime)

TABLE 54 Integration data (% of total surface area) of the differentpeaks observed in the SE-HPLC chromatograms of Polypeptide I stored at37° C. in different formulation buffers at all time points tested and incomparison with each control sample (each buffer). Phosphate pH 7Phosphate pH 7 Acetate pH 5.5 Acetate pH 5.5 Acetate pH 5.5 Histidine pH5.5 SE-HPLC Sample 50 mM NaCl 100 mM NaCl Phosphate pH 7 50 mM NaCl 100mM NaCl 10% Mannitol 50 mM NaCl % control 0 0 0 0 0 0 0 Prepeak 2 w 37°C. 5.6 6.9 1.3 4.6 6.3 2.3 5.5 3 w 37° C. 4.4 6.2 0.65 3.9 5.9 0.18 5.65 w 37° C. 13.7 15.8 3.9 11.5 14.2 1.22 14.0 10 w 37° C.  23.8 25.3 11.121.0 23.9 3.4 27.2 % Main control 100 100 100 100 100 100 100 peak 2 w37° C. 93.5 92.2 97.9 94.8 93.1 98.8 94.0 3 w 37° C. 93.7 92.0 95.2 95.092.8 96.9 93.4 5 w 37° C. 81.14 78.87 91.52 87.38 84.63 97.87 84.85 10 w37° C.  69.2 68.0 80.5 77.5 74.7 95.1 71.3 % control 0 0 0 0 0 0 0Postpeak1 2 w 37° C. 0 0 0 0 0 0 3 w 37° C. 0 0 0 0 0 0 5 w 37° C. 3.163.36 0 0 0 0 10 w 37° C.  3.7 3.5 0 0 0 0 % control 0 0 0 0 0 0Postpeak2 2 w 37° C. 0.23 0.27 0.19 0.23 0.26 0.19 0.19 3 w 37° C. 0.570.58 0.31 0.49 0.53 0.27 0.48 5 w 37° C. 0.41 0.47 0.27 0.37 0.39 0.250.45 10 w 37° C.  0.5 0.5 0.3 0.4 0.4 0.2 0.4 % control 0 0 0 0 0 0 0Postpeak3 2 w 37° C. 0.62 0.64 0.60 0.37 0.41 0.46 0.31 3 w 37° C. 1.151.25 1.07 0.52 0.64 0.61 0.49 5 w 37° C. 1.59 1.50 1.49 0.75 0.78 0.660.70 10 w 37° C.  2.7 2.6 3.1 1.1 1.0 1.3 1.1 Histidine pH 5.5 HistidinepH 5.5 Histidine pH 6 Histidine pH 6 Histidine pH 6 SE-HPLC Sample 100mM NaCl 10% Mannitol 50 mM NaCl 100 mM NaCl 10% Mannitol % control 0 0  0 0 0   Prepeak 2 w 37° C. 7.5 0.54 6.3 7.7 0.63 3 w 37° C. 7.9 0.34 7.08.6 0.39 5 w 37° C. 17.1 1.5  16.2 17.4 2.0  10 w 37° C.  27.8 5.4  26.827.0 7.3  % Main control 100 100*    100 100 100*    peak 2 w 37° C.92.1 98.8  93.1 91.5 96.7  3 w 37° C. 91.5 98.6  91.3 90.2 98.8  5 w 37°C. 81.73 97.49  82.22 81.19 96.76  10 w 37° C.  73.5 93.1  71.3 71.291.0  % control 0 0   0 0 0   Postpeak1 2 w 37° C. 0 0   0 0 0   3 w 37°C. 0 0   0 0 0   5 w 37° C. 0 0   0 0 0   10 w 37° C.  0 0   0 0 0   %control 0 0   0 0 0   Postpeak2 2 w 37° C. 0.17 0.19 0.20 0.23 0.18 3 w37° C. 0.55 0.27 0.54 0.5 0.27 5 w 37° C. 0.29 0.23 0.52 0.42 0.37 10 w37° C.  0.5 0.2  0.4 0.4 0.3  % control 0 0   0 0 0   Postpeak3 2 w 37°C. 0.26 0.37 0.40 0.58 0.53 3 w 37° C. 0.55 0.57 1.12 0.71 0.56 5 w 37°C. 0.88 0.78 1.06 0.99 0.87 10 w 37° C.  1.3 1.3  1.5 1.4 1.5 

The peak area of the prepeak increased over time but was reduced by theaddition of mannitol to the buffer (Table 54). The postpeaks between48-55 minutes elution time corresponded to degradation products (due toremaining proteolytic activity in sample). The relative area (%) ofthese peaks increased only slightly, implying that degradation wasrestricted to a minimum.

The prepeak represented the dimeric form of Polypeptide I. The peaksurface area of the prepeak increased with storage time (Table 54) andwas accompanied by a concomitant decrease in surface area of the mainpeak (Table 54). The propensity to form dimers was significantly lowerin the formulations containing 10% mannitol, which seemed to have apositive effect in suppressing the dimerization process. Note thesignificant lower amounts of dimers observed in the Acetate andHistidine buffers (pH 5.5) containing 10% mannitol (Table 54 and FIG.75). FIG. 75(A) summarizes the % surface area for the main peak in thedifferent buffers and at different time points when stored at 37° C.FIG. 75(B) summarizes the data for the % prepeak (dimer).

9.2 BIACORE Potency Analysis of the Polypeptide I Stored at 37° C.

The RANKL and HSA binding of Polypeptide I in stability samples storedfor 10 weeks at 37° C. was compared with the activity of the unstressedreference batch using BIACORE analysis. RANKL or HSA was immobilized onthe BIACORE chip (amine coupling using the BIACORE amine coupling kit).After a preconditioning step of 5 injections of Polypeptide I, allsamples were diluted to 2.5 nM in triplicate and analyzed on the chip.Slopes were determined using the general fit method and the linear fitmodel (BIAevaluation software). To determine the initial binding rate(IBR), the slope between 5 s and 30 s was selected. The values of theseslopes were transferred in excel and the percentage activity/potencycompared to the Polypeptide I reference material was determined. BIACOREpotency is thus expressed as relative potency compared to the referencematerials. The relative potencies are given in Table 55 and areexpressed as % activity compared to reference batch.

After 10 weeks of storage at 37° C. the relative potency of PolypeptideI for binding RANKL had dropped to 70-80% in the different buffers(Table 55). In histidine, pH 6+10% mannitol, the activity remained thehighest (87.4%). The higher the NaCl concentration in the buffer, thelower the relative potency in the sample (compare the values obtained inbuffers with 50 mM NaCl and 100 mM NaCl in Table 55).

TABLE 55 Relative potencies of the HSA and RANKL binding moieties ofPolypeptide I after 10 weeks at 37° C. as measured by BIACORE analysis.Relative potency Buffer RANKL HSA Phosphate + SO mM NaCl, pH 7 81.0 57.4Phosphate + 100 mM NaCl, pH 7 78.6 56.6 Phosphate + 10% Mannitol, pH 776.3 66.8 Acetate + 50 mM NaCl, pH 5.5 80.1 63.0 Acetate + 100 mM NaCl,pH 5.5 78.0 59.0 Acetate + 10% Mannitol, pH 5.5 80.9 79.4 Histidine + 50mM NaCl, pH 5.5 80.2 59.7 Histidine + 100 mM NaCl, pH 5.5 73.1 55.0Histidine + 10% Mannitol, pH 5.5 75.2 73.6 Histidine + 50 mM NaCl, pH 679.1 59.3 Histidine + 100 mM NaCl, pH 6 78.3 57.5 Histidine + 10%Mannitol, pH 6 87.4 83.4

The relative potency for HSA binding had dropped more compared to theactivity for RANKL binding after 10 weeks storage at 37° C. Thisdecrease in activity however was less significant in themannitol-containing buffers than in the NaCl-containing buffers. Asobserved for RANKL binding, the percentage activity on HSA decreasedwith increasing concentrations of NaCl in the different buffers.

Example 10 Tm Determination of Polypeptides J and K

Polypeptide J (SEQ ID NO: 18) is a bispecific Nanobody consisting of twohumanized variable domains of a heavy-chain llama antibody, one bindingto IL-6R, the other one (Alb11) binding to HSA. The trivalent bispecificPolypeptide K (SEQ ID NO: 19) consists of two identical subunits thatare specific for IL-6R while the third subunit binds to HSA.

The polypeptides were expressed in Pichia pastoris. Concentration of thepolypeptide and buffer switch to PBS or other formulation buffer wasdone via UF/DF (Sartorius Hydrosart SARTOCON Slice 200, 10 kDa) ordialysis. A final filtration was carried out at 0.22 μm.

The melting temperature (Tm) in different buffers was determined usingthe fluorescence-based thermal shift assay. The thermal shift assay orTSA can be performed in 96-well plate in a Q-PCR device to evaluate theeffect of buffer couple, ionic strength, pH and excipients on thethermal stability of proteins. The assay results in a Tm value that isindicative for the thermal stability in the tested buffers. Briefly, theassay follows the signal changes of a fluorescence dye, such as SYPROOrange, while the protein undergoes thermal unfolding. When SYPRO Orangeis added to a properly folded protein solution, it is exposed in anaqueous environment and its fluorescence signal is quenched. When thetemperature rises, the protein undergoes thermal unfolding and exposesits hydrophobic core region. SYPRO Orange then binds to the hydrophobicregions, unquenches which results in the increase of the fluorescencesignal.

The Tm was assessed for Polypeptide J and Polypeptide K in differentbuffers, excipients and combinations thereof using the TSA assay. Theobtained Tm values are displayed graphically in FIGS. 76 to 80. In allconditions tested, the Tm values were slightly higher for Polypeptide Jthan Polypeptide K. The excipients tested (mannitol, sucrose andglycine) had a similar effect on the Tm values of Polypeptide J andPolypeptide K. All excipients tested appeared to have a stabilizingeffect on Polypeptide J and Polypeptide K, since the meltingtemperatures increased with increasing excipient concentration. Thehighest Tm values were obtained in buffers containing 7.5% mannitol or5% sucrose.

Example 11 Storage Stability Study of Polypeptides J and K at 37° C.

An initial storage stability study was performed to get a generalunderstanding of the stability of Polypeptides J, K and L and todetermine if adding mannitol in the formulation buffer has a beneficialeffect in minimizing the formation of potential dimers, as was observedfor Polypeptide I (see Example 9). The trivalent bispecific PolypeptideL (SEQ ID NO: 20) consists of two identical subunits that are specificfor IL-6R while the third subunit binds to HSA.

The three Polypeptides were formulated in different buffers (Table 56)at a concentration of 10 mg/mL (Polypeptide J), 7.1 mg/mL (PolypeptideK) and 10.3 mg/mL (Polypeptide L).

TABLE 56 Overview of the different formulation buffers used in initialstability testing of Polypeptide J, Polypeptide K and Polypeptide L.Condition Buffer [NaCl] Mannitol 1 PBS  0 mM 0% 2 PBS  0 mM 5% 3 10 mMNaH₂PO₄•2H₂O, pH 7 100 mM 0% 4 10 mM NaH₂PO₄•2H₂O, pH 7 100 mM 5% 5 10mM Na-acetate, pH 5.5 100 mM 0% 6 10 mM Na-acetate, pH 5.5 100 mM 5% 720 mM L-histidine, pH 6 100 mM 0% 8 20 mM L-histidine, pH 6 100 mM 5%

The stability of the different samples was assessed in acceleratedstress conditions at 37° C. Samples were analyzed after 1 week usingSE-HPLC. Selected samples of Polypeptides J and K were also analyzedafter 3 weeks of storage. The SE-HPLC assay consisted of a pre-packedPhenomenex BIOSEP SEC S2000 column, a mobile phase consisting of KCl,NaCl and phosphate buffer pH 7.2 (D-PBS) and UV detection at 280 nm. Therelative amount of specific protein impurity was expressed as area %,and was calculated by dividing the peak area corresponding to thespecific protein or protein impurity by the total integrated area. Themethod can resolve and quantify the relative amounts of intact materialand product related impurities such as aggregates and degradationfragments.

For both Polypeptides, prolonged storage at 37° C. resulted in theformation of prepeaks and some minor postpeaks. The postpeaks probablycorresponded to degradation products (due to remaining proteolyticactivity in sample). The surface area of these postpeaks remained verylow, suggesting only minimal degradation after 3 weeks at 37° C.

Both Polypeptides had a strong tendency to form dimers/oligomers(aggregates), which were visible as prepeak(s) in the chromatograms ofthe SE-HPLC analysis. An example chromatogram is shown in FIG. 81. Thepeak area of the prepeak increased significantly over time (representedas % aggregates in FIG. 82) and was accompanied by a concomitantdecrease in surface area of the main peak. The lowest amounts ofoligomers were observed in the mannitol-containing formulations.

Example 12 Storage Stability Study of Polypeptide J at 5° C. and 37° C.

An overview of the different formulation buffers and methods used instability testing of Polypeptide J is given in Table 57 and Table 58,respectively.

TABLE 57 Overview of the different formulation buffers used in stabilitytesting of Polypeptide J. Concen- tration % % % mM Buff- Poly- TweenMan- Su- Gly- er peptide J Buffer 80 nitol crose cine 1 10 mg/mL 20 mML-histidine / / / / 2 10 mg/mL 20 mM L-histidine 0.01 / / / 3 10 mg/mL20 mM L-histidine 0.05 / / / 4 10 mg/mL 20 mM L-histidine 0.05 5 / / 510 mg/mL 20 mM L-histidine 0.05 5 / 200 6 10 mg/mL 20 mM L-histidine0.05 2.5 / 100 7 10 mg/mL 20 mM L-histidine 0.05 / 10  / 8 10 mg/mL 20mM L-histidine 0.05 / / 200 9 10 mg/mL 20 mM L-histidine 0.05 / 5 100 1010 mg/mL 20 mM L-histidine 0.05 2.5 5 / 11 10 mg/mL 20 mM L-histidine /2.5 5 100 12 10 mg/mL 20 mM L-histidine 0.05 2.5 5 100

TABLE 58 Methods used for assessing the stability of Polypeptide J atdifferent time points (represented as x weeks or w) after storage at 5°C. and 37° C. Stress condition Method Purpose Ref. material 5° C. 37° C.A280 Content 0 w 1, 2 and 5 w 1, 2, 3 and 5 w Appearance Precipitation 0w 1, 2 and 5 w 1, 2, 3 and 5 w RPC Purity/ 0 w 1, 2 and 5 w 1, 2, 3 and5 w variants SEC Purity/ 0 w 1, 2 and 5 w 1, 2, 3 and 5 w aggregation/ 6months 6 months hydrolysis BIACORE Potency 0 w 5 w 5 w (HSA binding)Osmolality Charac- 0 w / / teristic

Samples of the reference material (0 weeks) and samples stored for up to6 months at 5° C. and 37° C. were analyzed using SE-HPLC. No differenceswere observed between the SE-HPLC profiles of the reference samples (at0 weeks) and the samples stored for up to 5 weeks at 5° C. SE-HPLCanalysis of the samples stored for 6 months at 5° C. did not showincrease in area % of the prepeaks, meaning that no oligomers wereformed under these storage conditions, not even in the formulationcontaining only 20 mM L-histidine, pH 6.5 i.e. without TWEEN(polysorbate) 80 or any excipient (data not shown).

Prolonged storage at 37° C. resulted in the formation of prepeaks andsome minor postpeaks. The postpeaks probably corresponded to degradationproducts (due to remaining proteolytic activity in sample). The relativearea (%) of these peaks increased only slightly, implying thatdegradation was restricted to a minimum. The other peaks visible in thechromatograms were background peaks arising from the buffer components.

The peak area of the prepeaks increased significantly over time (FIG. 83and FIG. 84). Given the relative position of the prepeaks to the mainpeak, the prepeaks most likely represented dimeric or oligomeric forms(aggregates) of Polypeptide J. The peak surface area of the prepeakincreased with storage time and was accompanied by a concomitantdecrease in surface area of the main peak.

An important observation was that the propensity to formdimers/oligomers was buffer-dependent: the propensity to oligomerize wassignificantly lower in the mannitol- and sucrose-containingformulations. Glycine appeared not to have such a positive effect inpreventing the oligomerization process. TWEEN (polysorbate) 80 had noinhibitory effect on the formation of oligomers.

In the samples stored for 6 months at 37° C., the lowest % of oligomerswas found in the formulation containing 10% sucrose, again corroboratingthe stabilizing effect of sucrose on Polypeptide J (Table 59).

Example 13 Storage Stability Study of Polypeptide J at −70° C., −20° C.5° C. 25° C. and 37° C.

Polypeptide J was formulated at 10 mg/mL in the 10 different buffersshown in Table 60, stored at −70° C., −20° C., +5° C. and 37° C. for 8weeks and for 1 week +25° C. Stability samples were analyzed usingSE-HPLC. Selected samples were also analyzed using BIACORE (HSA binding)and potency assays (HSA and IL-6R).

TABLE 59 Overview of the SE-HPLC integration results after storage for 6months at 37° C. Buffer % pre peak 1 % pre peak 2 % main peak % postpeak Ref  0.52 0.17 99.3 0   Buffer 1 ND ND ND ND Buffer 2 20.4 2.1 73.44.1 Buffer 3 ND ND ND ND Buffer 4 18.1 17 76.0 4.2 Buffer 5 22.2 2.071.4 4.4 Buffer 6 21.4 1.7 72.7 4.2 Buffer 7 15.1 0 80.5 4.4 Buffer 821.1 2.4 72.0 4.5 Buffer 9 16.7 2.7 76.3 4.3 Buffer 10 15.8 1.9 77.9 4.4Buffer 11 17.5 2.0 76.4 4.2 Buffer 12 16.8 3.3 75.7 4.2

TABLE 60 Overview of the different formulation buffers tested in thestability study. Nr. Conc. Buffer Mannitol Sucrose Trehalose GlycineTween-80 1 10 mg/mL 15 mM L-histidine, pH 6.5  5% 0.01% 2 10 mg/mL 15 mML-histidine, pH 6.5 10% 0.01% 3 10 mg/mL 15 mM L-histidine, pH 6.5 10%0.01% 4 10 mg/mL 15 mM L-histidine, pH 6.5 7.5%  0.35% 0.01% 5 10 mg/mL15 mM L-histidine, pH 6.5 2.5%  5% 0.01% 6 10 mg/mL 15 mM phosphate, pH6.5  5% 0.01% 7 10 mg/mL 15 mM phosphate, pH 6.5 10% 0.01% 8 10 mg/mL 15mM phosphate, pH 6.5 10% 0.01% 9 10 mg/mL 15 mM phosphate, pH 6.5 7.5% 0.35% 0.01% 10 10 mg/mL 15 mM phosphate, pH 6.5 2.5%  5% 0.01%

13.1 Storage for 8 Weeks at −70° C., −20° C., 5° C. and 1 Week at 25° C.

Polypeptide J was shown to be stable after storage for 8 weeks at −70°C., −20° C., 5° C. and for 1 week at 25° C. in all 10 buffers tested. Nosignificant differences were observed in potency, and SE-HPLC profilesbetween the reference material and the 10 different storage samples(data not shown).

132. Storage for 8 Weeks at 37° C. SE-HPLC

Prolonged storage at 37° C. resulted in the time-dependent formation ofa postpeak and prepeak. The postpeak has a retention time between 22 and23 minutes and most likely corresponded to Polypeptide J degradationfragments. The surface area of this peak however remained low(approximately 2%), suggesting only minimal degradation after 8 weeks at37° C. The other postpeaks visible in the chromatograms were backgroundpeaks arising from the buffer components.

The SE-HPLC profile of Polypeptide J at time point 0 weeks included amain peak and two minor prepeaks, which were not completelybaseline-resolved. The surface area of the prepeaks increased over time(FIG. 85) and was accompanied by a concomitant decrease in surface areaof the main peak. Given the relative position and heterogeneity of theprepeaks, they most likely represented dimeric and/or oligomeric formsof Polypeptide J. Because of this heterogeneity and the decreasingresolution between the prepeaks over time, the peaks were for simplicityintegrated as a single peak.

An important observation was that the propensity to formdimers/oligomers was buffer-dependent: about 2-fold less oligomers werebeing formed in L-histidine buffer compared to phosphate buffer (FIG.86, FIG. 87). The lowest amount of oligomers was observed in thetrehalose-containing formulation, followed by the sucrose-containingformulation. The presence of a non-reducing sugar suppressed the extentof Polypeptide J oligomerization considerably.

Potency Assay and BIACORE

The potency of the samples stored for 8 weeks at 37° C. in buffers 1-5was determined relative to an unstressed reference batch using anHSA-binding ELISA and an inhibition ELISA for IL-6R (Table 61).

In the ELISA based potency assay for HSA binding, human serum albumin(HSA) was immobilized onto a multiwell MAXISORP ELISA plate byadsorption. After blocking excess binding sites on the plates withSuperblock T20 (PBS) blocking buffer, a dilution series of test andreference samples was applied on the plate. Bound Polypeptide wassubsequently detected using a bivalent anti-Nanobody Nanobody directlyconjugated to horseradish peroxidase (HRP). In the presence of H₂O₂ HRPcatalyzes a chemical reaction with Tetramethylbenzidine (es TMB) whichresults in the formation of a color. The reaction was stopped by adding1N HCl. The optical density of the color was measured at 450 nm.

In the ELISA based potency assay for IL-6R binding, for the reference,control and test samples, different dilutions of the Polypeptides wereprepared. These dilutions were pre-incubated with a constant amount of100 ng/mL IL-6, followed by the addition of 4 ng/mL soluble IL-6R.Subsequently, this mixture was transferred to a microtiter plate coatedwith a non neutralizing anti-IL-6R Nanobody. After washing, residualbound IL-6 was detected with biotinylated anti-human IL-6 monoclonalantibody, followed by HRP-labeled streptavidin detection. In thepresence of H₂O₂ HRP catalyzes a chemical reaction withTetramethylbenzidine (es TMB) which results in the formation of a color.The reaction was stopped by adding 1N HCl. The optical density of thecolor was measured at 450 nm. The relative potency of the test samplescompared to the reference sample was analyzed by use of PLA 2.0Software.

The HSA binding functionality of the samples stored in buffers 1-10 wasalso analyzed using BIACORE (Table 62). For the affinity measurement onBIACORE, a chip was first immobilized with HSA (amine coupling using theBIACORE amine coupling kit). After a preconditioning step of 5injections of the Polypeptide J, all samples were diluted to 2.5 nM intriplicate and analyzed on the chip. Quality control of the chips usingthe reference sample was included in the experiment to detect any lossof activity or decrease in response (deterioration of the chip). Slopeswere determined using the general fit method and the linear fit model(BIAevaluation software). To determine the initial binding rate (IBR),the slope between 5 s and 30 s was selected. The values of these slopeswere transferred in excel and the percentage activity compared to thereference was determined.

Samples formulated in the same buffers and stored at −70° C. wereincluded as the reference molecules.

TABLE 61 Relative potency of Polypeptide J after 8 weeks at +37° C.compared to a reference sample. Buffer HSA IL-6R 1 1.080 (0.954-1.223)1.153 (0.957-1.389) 2 0.975 (0.887-1.072) 0.980 (0.760-1.263) 3 1.038(0.952-1.132) 1.117 (0.910-1.372) 4 1.182 (1.074-1.300) 1.061(0.908-1.240) 5 1.080 (1.004-1.161) 1.082 (0.925-1.266)

TABLE 62 Summary of the BIACORE results for HSA binding of the stabilitysamples stored for 8 weeks at 37° C., expressed as % activity comparedto the equivalent sample stored at −70° C. Buffer % activity compared toreference 1 97.5 2 93.2 3 92.5 4 83.9 5 101.9 6 92.2 7 89.4 8 99.0 984.3 10 89.6

Whereas the potency assays showed comparable HSA and IL-6R bindingpotencies between the stability samples and the reference material,BIACORE analysis demonstrated some differences in HSA bindingactivities. A functionality loss of approximately 16% was observed inthe buffers containing a combination of sucrose and glycine (buffer 4and 9). Formulations containing either mannitol, sucrose or trehaloseshowed an activity between 90 and 100% after storage for 8 weeks at 37°C.

TABLE A Sequence Listings SEQ ID Code NO: Sequence Polypeptide A  7EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS Polypeptide B  8EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTL VTVSSPolypeptide C  9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFITSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTL VTVSSPolypeptide D 10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNITYLQMNSLRPEDTAVYYCAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYIKKVPQVSTRTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Polypeptide E 11EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIGWFRQAPGKGREGVSGISSSDGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYKAAEPPDSSWYLDGSPEFFKYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Polypeptide F 12AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQ VTVSS Ligand A13 DISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTHHHHHH CDR3 and FR4 14GGSLSRSSQGTLVTVSS of polypeptide B Polypeptide G 15EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQ VTVSSPolypeptide H 16 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQ VTVSSPolypeptide I 17 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTESSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS Polypeptide J 18EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Polypeptide K 19EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Polypeptide L 20EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITIESDYDLGRRYWGQGTLVTVSS

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All of the references described herein are incorporated by reference, inparticular for the teaching that is referenced hereinabove.

1. A formulation comprising an aqueous carrier having a pH of 5.5 to 8.0and a polypeptide comprising one or more single variable domains at aconcentration of 1 mg/mL to 200 mg/mL, said formulation being formulatedfor administration to a human subject and said formulation furthercomprising: a histidine pH 6.0-6.5 buffer at a concentration of 10 mM to100 mM, wherein said formulation has an inorganic salt concentration of150 mM or lower and wherein the polypeptide comprises at least onesingle variable domain that specifically binds human serum albumin(HSA).
 2. The formulation of claim 1, that does not contain anyinorganic salt.
 3. The formulation of claim 1, wherein the concentrationof polypeptide is about 1 to 200 mg/ml or more, about 5 to 100 mg/mL ormore, about 5 to 50 mg/mL or more, about 5 to 30 mg/mL or more, around 5mg/mL, around 10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40mg/mL, around 50 mg/mL, around 60 mg/mL, around 70 mg/mL, around 80mg/mL, around 90 mg/mL, around 100 mg/mL, or around 150 mg/mL.
 4. Theformulation of claim 1, wherein the polypeptide comprises two or moresingle variable domains, or two or three single variable domains.
 5. Theformulation of claim 1, wherein the at least one single variable domainthat specifically binds HSA has following sequence: (SEQ ID NO: 21)EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSS.


6. The formulation of claim 1, wherein the polypeptide has a solubilityof at least 20 mg/mL, 50 mg/mL or more, 90 mg/mL or more, 120 mg/mL ormore, 150 mg/mL or more, or even 200 mg/mL or more, as determined by thepolyethylene glycol (PEG) exclusion method or by a concentrationexperiment; the polypeptide has a melting temperature of at least 59° C.or more, at least 60° C. or more, at least 61° C. or more, at least 62°C. or more, or at least 63° C. or more as measured by the thermal shiftassay (TSA) and/or differential scanning calorimetry (DSC); noparticulates are present as measured by OD320/OD280 and/or elastic lightscattering; less than 10% of the polypeptide forms pyroglutamate at theN-terminal glutamic acid during storage at a temperature of 37±5° C. upto at least 2 weeks, at least 3 weeks, at least 5 weeks, at least 8weeks, at least 10 weeks, at least 3 months, at least 6 months, at least1 year, at least 1.5 years or at least 2 years, the % of pyroglutamateas measured by Reversed Phase High Performance Liquid Chromatography(RP-HPLC); less than 10% of the polypeptide forms dimers during storageat a temperature of 37±5° C. up to at least 2 weeks, at least 3 weeks,at least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3months, at least 6 months, at least 1 year, at least 1.5 year or atleast 2 years, the % of dimers as measured by Size Exclusion HighPerformance Liquid Chromatography (SE-HPLC); at least 80% of thepolypeptide retains its binding activity to at least one of its targetsafter storage at 37±5° C. up to at least 2 week, at least 3 weeks, atleast 5 weeks, at least 2 months, at least 6 months, at least 1 year,1.5 year or even 2 years or more compared to the binding activity priorto storage, said binding activity as measured by enzyme-linkedimmunosorbent assay (ELISA) and/or Surface Plasmon Resonance; and/or thepolypeptide is stable during mechanical stress.
 7. (canceled)
 8. Theformulation of claim 1, further comprising an excipient at aconcentration of 1% to 20% (w:v).
 9. (canceled)
 10. The formulation ofclaim 1, wherein the histidine buffer has a concentration of 10 to 50mM, 10 to 20 mM, 10 mM or 15 mM.
 11. The formulation of claim 8, whereinthe excipient is a saccharide, a non-reducing sugar and/or polyol. 12.The formulation of claim 11, wherein the excipient is selected from thegroup consisting of mannitol and sucrose.
 13. The formulation of claim8, wherein the excipient has a concentration of 2.5% to 15% (w:v), 5% to10% (w:v), 5% (w:v), 7.5% (w:v), 8% (w:v) or 10% (w:v).
 14. Theformulation of claim 1, further comprising a surfactant at aconcentration of 0.001% to 1% (v:v) selected from polysorbate 80,polysorbate 20 or a poloxamer.
 15. The formulation of claim 14, whereinthe surfactant has a concentration of 0.01% to 0.1% (v:v), 0.01% to0.05% (v:v), 0.01% (v:v) or 0.005% (v:v).
 16. The formulation of claim1, comprising: a) a histidine pH 6.5 or pH 6.0 buffer at a concentrationof 10 mM to 100 mM; b) an excipient at a concentration of 1% to 20%(w:v); and c) a surfactant at a concentration of 0.001% to 1% (v:v)selected from polysorbate 80, polysorbate 20 or a poloxamer. 17.-20.(canceled)
 21. A method for the preparation of a formulation of claim 1,at least comprising the step of concentrating the polypeptide andexchanging it with the selected buffer and/or excipient.
 22. A sealedcontainer containing a formulation according to claim
 1. 23. Apharmaceutical unit dosage form suitable for parenteral administrationto a human, comprising a formulation according to claim 1 in a suitablecontainer.
 24. A kit comprising one or more of the sealed containersaccording to claim 22 and instructions for use of the formulation. 25.(canceled)
 26. Method for prevention and/or treatment of one or morediseases and/or disorders, comprising administering to a subject in needthereof a formulation according to claim
 1. 27. (canceled)
 28. A kitcomprising one or more of the pharmaceutical unit dosage forms accordingto claim 23, and instructions for use of the formulation.